Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 10, pp. 1660-1680 © The Author(s) 2024. This article is an open access publication.

1660

REVIEW

ω-Amidase and Its Substrate α-Ketoglutaramate

(the α-Keto Acid Analogue of Glutamine)

as Biomarkers in Health and Disease

Arthur J. L. Cooper

1,a#

and Travis T. Denton

2,3,4,5,b,c

Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA

LiT Biosciences, 120 N Pine ST, Ste 242, Spokane, WA 99202-5029, USA

Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences,

Washington State University Health Sciences Spokane, Spokane, WA, USA

Department of Translational Medicine and Physiology, Elson S. Floyd College of Medicine,

Washington State University Health Sciences Spokane, Spokane, WA, USA

Steve Gleason Institute for Neuroscience, Washington State University Health Sciences Spokane, Spokane, WA, USA

e-mail: arthur_cooper@nymc.edu; ORCID 0000-0002-9143-8504

e-mail: travis.denton@litbiosciencesllc.com; ORCID 0000-0002-1222-2538

e-mail: travis.denton@wsu.edu

Received July 12, 2024

Revised September 10, 2024

Accepted September 15, 2024

Abstract— A large literature exists on the biochemistry, chemistry, metabolism, and clinical importance of the

α-keto acid analogues of many amino acids. However, although glutamine is the most abundant amino acid

in human tissues, and transamination of glutamine to its α-keto acid analogue (α-ketoglutaramate; KGM) was

described more than seventy years ago, little information is available on the biological importance of KGM.

Herein, we summarize the metabolic importance of KGM as an intermediate in the glutamine transaminase –

ω-amidase (GTωA) pathway for the conversion of glutamine to anaplerotic α-ketoglutarate. We describe some

properties ofKGM, notably its occurrence as a lactam (2-hydroxy-5-oxoproline; 99.7% at pH7.2), and its presence

in normal tissues and body fluids. We note that the concentration of KGM is elevated in the cerebrospinal fluid

of liver disease patients and that the urinary KGM/creatinine ratio is elevated in patients with an inborn error

of the urea cycle and in patients with citrin deficiency. Recently, of the 607 urinary metabolites measured in a

kidney disease study, KGM was noted to be one of five metabolites that was most significantly associated with

uromodulin (a potential biomarker for tubular functional mass). Finally, we note that KGM is an intermediate

in the breakdown of nicotine in certain organisms and is an important factor in nitrogen homeostasis in some

microorganisms and plants. In conclusion, we suggest that biochemists and clinicians should consider KGM as

(i) akey intermediate in nitrogen metabolism in all branches of life, and (ii) a biomarker, along with ω-amidase,

inseveral diseases.

DOI: 10.1134/S000629792410002X

Keywords: ω-amidase, ammonia, anaplerosis, glutamine, glutaminase 1, glutaminase 2, glutamine addiction, glu-

tamine transaminases, 2-hydroxy-5-oxoproline, hyperammonemia, α-ketoglutarate, α-ketoglutaramate, methi-

onine, methionine salvage pathway, transamination

* To whom correspondence should be addressed.

# Deceased.

DEDICATION

This article is dedicated to my long-time friend

and mentor, Dr. Arthur J. L. Cooper. We suddenly lost

Dr. Cooper on May 30, 2024, at the age of 78, during

the time he and I were in the process of submitting

this article together. Arthur was, undeniably, one of

the world’s foremost experts in glutamine metabo-

lism, as well as countless other areas of biochemistry

and neuroscience. Arthur spent much time in the final

years of his life seeking to raise scientific awareness

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1661

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

about the Glutamine Transaminase-omega-Amidase

(GTωA) pathway. It is in Arthur’s memory that I pres-

ent this final publication highlighting the GTωA path-

way, co-authored with Arthur J. L. Cooper. You will

never be forgotten, my friend!

INTRODUCTION. THE DISCOVERY

OF ENZYME-CATALYZED TRANSAMINATION

A discussion of the discovery of enzyme-catalyzed

transamination provides a background for the discov-

ery of glutamine transamination. Enzyme-catalyzed

transamination [Eq.(1)] was discovered by Alexander E.

Braunstein and coworkers in the 1930s. For a review of

this work and its metabolic importance see [1]. Braun-

stein coined the word “umaminierung” (i.e., transami-

nation) to describe this process. Enzymes that catalyze

transamination reactions were originally referred to as

transaminases. However, currently, the more common

term for transaminases is aminotransferases. In this re-

view we will use the original term transaminase where

appropriate. The first transamination reactions discov-

ered by Braunstein and colleagues were between

-glu-

tamate and pyruvate [Eq.(2)] and between

-glutamate

and oxaloacetate [Eq. (3)]. The enzymes that catalyze

these reactions are often referred to as glutamate py-

ruvate transaminase (GPT) and glutamate oxaloacetate

transaminase (GOT), respectively, especially among cli-

nicians. The recommended Enzyme Commission terms

for these enzymes are alanine aminotransferase and

aspartate aminotransferase, respectively, where it is

understood that L-glutamate is the amino acid substrate

in the forward direction. Here, we use the abbrevia-

tions AlaAT and AspAT, respectively, for these enzymes.

L-Amino acid (1) + α-keto acid (2) ⇆

α-keto acid (1) +

L-amino acid (2)

(1)

L-Glutamate + pyruvate ⇆

α-ketoglutarate +

L-alanine

(2)

L-Glutamate + oxaloacetate ⇆

α-ketoglutarate +

L-aspartate

(3)

Mammalian tissues contain two isozymes of

AspAT – a mitochondrial form (mitAspAT) and a cy-

tosolic form (cytAspAT). Each is a homodimer. The pig

heart cytosolic and mitochondrial isozyme monomers

contain 412 and 401 residues, respectively ([1] and ref-

erences cited therein). In 1973, after enormous effort,

Braunstein and coworkers sequenced pig heart cytoso-

lic AspAT monomer [2] – at that time, it was the third

largest protein to have been sequenced! Braunstein

was the first to emphasize the importance of gluta-

mate/α-ketoglutarate-linked aminotransferases [Eq.(4)]

coupled to the glutamate dehydrogenase (GDH) reac-

tion [Eq.(5)] in generating ammonia from many amino

acids, or for assimilating ammonia nitrogen into many

amino acids in mammalian issues. He termed these

processes transdeamination and transreamination, re-

spectively [3]. The transdeamination reaction [forward

direction of Eq.(6)] is especially important in directing

nitrogen from the catabolism of many of the common

amino acids toward ammonia to be used for urea syn-

thesis in the liver [4]. Esmond Snell (e.g.,[5, 6]), Alton

Meister (e.g., [7, 8]) and their colleagues established

pyridoxal 5′-phosphate (PLP) as the cofactor involved

in transamination reactions.

L-Amino acid + α-ketoglutarate ⇆

α-keto acid +

L-glutamate

(4)

L-Glutamate + H

O + NAD

⇆

α-ketoglutarate + NADH +

(5)

Net:

L-Amino acid + H

O + NAD

⇆

α-keto acid + NADH +

(6)

Following the pioneering work of Braunstein and

colleagues it was soon established that transamination

in mammals is a major step in the metabolism of, for

example, aspartate, branched chain amino acids, γ-am-

inobutyric acid (GABA), tyrosine and phosphoserine.

However, discussion of transaminases involved in the

metabolism of these amino acids is beyond the scope

of the present review.

DISCOVERY AND PROPERTIES

OF L-GLUTAMINE TRANSAMINASES

Discovery. In the late 1940s, Alton Meister and col-

leagues at the National Institutes of Health discovered

transamination of L-glutamine to α-ketoglutaramate

(KGM) [Eq.(7)]. They also discovered an enzyme that

they named ω-amidase, which catalyzes the hydrolysis

of KGM to α-ketoglutarate and ammonia [Eq.(8)]. The

net reaction resulting from the combination of gluta-

mine transaminase and ω-amidase [Eq.(9)] was named

the glutaminaseII pathway [9-12].

L-Glutamine + α-keto acid ⇆

α-ketoglutaramate (KGM) +

L-amino acid

(7)

KGM + H

O → α-ketoglutarate +

(8)

Net:

L-Glutamine + α-keto acid + H

O →

α-ketoglutarate +

L-amino acid +

(9)

For a description of the background leading to

the discovery of the glutaminase II pathway see [13].

COOPER, DENTON1662

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Fig.1. The glutamine transaminase-ω-amidase (GTωA) pathway. Glutamine transamination with a suitable α-keto acid sub-

strate is catalyzed by at least two glutamine transaminases in mammalian tissues– glutamine transaminaseK (GTK) and glu-

tamine transaminase L (GTL). The resulting α-keto acid (α-ketoglutaramate; KGM) is in equilibrium with a cyclic lactam form

(2-hydroxy- 5-oxoproline) [16, 17]. Under physiological conditions, the lactam predominates (~99.7%) over the open chain form

(~0.3%; ω-amidase substrate) [16, 17]. [Throughout this review, the term KGM is understood to be the sum of open-chain form

plus lactam, unless specified otherwise.] Note that although the transamination of glutamine is freely reversible (see[13]),

the pathway is irreversibly pulled in the direction of glutamine transamination by cyclization of KGM and by the action

ofω-amidase. Note also that not all enzyme reactants and products are depicted.

In order to prevent confusion between the name glu-

taminaseII and the “true” glutaminase (GLS2) we have

recently renamed the glutaminase II pathway as the

GTωA pathway [13]. The GTωA pathway is depicted

inFig.1.

Purification of glutamine transaminases. Meis-

ter and colleagues partially purified a glutamine trans-

aminase from rat liver and showed that the enzyme

exhibits wide specificity toward α-keto acids [9, 10].

Subsequently, Cooper and Meister purified the rat

liver enzyme to homogeneity and showed that it is

a homodimer, and PLP dependent [14]. The enzyme

was also shown to exhibit broad

L-amino acid speci-

ficity [14]. Cooper and Meister later showed that rat

tissues contain an additional glutamine transaminase

that is prominent in kidney and that they named gluta-

mine transaminaseK, to distinguish it from glutamine

transaminase L (GTL) – the predominant form in rat

liver [15]. Like the K-isozyme the

L-isozyme is a ho-

modimer and also has broad amino acid and α-keto

acid specificity.

These authors also noted that the activities are

present in cytosolic and mitochondrial fractions [15].

This was verified for GTK by Malherbe etal. [18] who

showed that the N-terminus of the rat enzyme possess-

es a 32 amino acid mitochondrial targeting sequence

that allows entry into the mitochondria; removal of

this sequence ensures a competing cytosolic location

[18]. The biological basis for the evolution of two gluta-

mine transaminases with such overlapping specificity

is currently unknown.

Nomenclature of glutamine transaminases and

subunit composition. As noted in [13], owing to the

broad substrate specificity of the

-glutamine transam-

inases, it is probable that some preparations of phe-

nylalanine, serine, histidine and aminoadipate trans-

aminases reported in the literature can be ascribed

L-glutamine transaminases. Of special note is the

finding that two kynurenine aminotransferase (KAT)

isozymes that have been described in the scientific lit-

erature, namely KAT1 and KAT3, are identical to GTK,

and GTL, respectively [13, 19-21]. In addition, KAT2

(aminoadipate aminotransferase) has some glutamine

transaminase activity [22].

Human GTK, which is annotated as KYAT1 in

UniProt, is a homodimer [23]. Three isoforms of hu-

man GTK/KYAT1, produced by alternative splicing, are

predicted in this database, namely Q16773-1 (canoni-

cal form; 422 amino acid residues; subunit molecular

mass 47,875 Da), Q16773-2 (canonical form missing res-

idues 68-117), and Q16773-3 (different sequence from

the canonical form at residues 230-250 and missing

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1663

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

residues 251-422). The biological roles of Q16773-2

and3 are unknown.

Human GTL, which is annotated as KYAT3 in

UniProt, is also a homodimer [24] and is predicted to

exist as three alternatively spliced isoforms. Isoform1

(Q6YP21-1) is the canonical isoform (length 454 ami-

no acid residues; subunit molecular mass 51,400 Da)

and contains a 34 amino acid mitochondrial targeting

sequence. Isoform 3 (Q6YP21-3) lacks residues 1-34

of the canonical isoform. Isoforms 1 and 3 presum-

ably account for the presence of the enzyme in the

mitochondria and cytosol, respectively. In addition, a

third isoform (Q6YP21-2) is greatly truncated (missing

residues 168-454) and contains an altered sequence

152-167. The function of this polypeptide is unknown.

A relatively large amount of work has been car-

ried out on the four KATs described in the scientific

literature (i.e., KATs 1, 2, 3, and 4) in part because

the product of kynurenine transamination (i.e., ky-

nurenate) is a neuromodulator (e.g.,[25] and referenc-

es cited therein). According to[25], kynurenate is “an

endogenous antagonist of α7 nicotinic acetylcholine

and excitatory amino acid receptors, regulates gluta-

matergic, GABAergic, cholinergic and dopaminergic

neurotransmission in several regions of the rodent

brain”. The contribution of GTK/KAT1 and GTL/KAT3 to

the formation of cerebral neuromodulator kynurenate

is debatable and KAT2 is now regarded as the probable

main contributor to brain kynurenate formation [26].

Moreover, among the amino acids tested as substrates

for both GTK/KAT1 and GTL/KAT3, the highest catalytic

efficiency (V

max

) occurs with L-glutamine. The cata-

lytic efficiency is much lower for kynurenine [19, 20].

In addition, L-glutamine is the most abundant amino

acid in human tissues and its concentration is estimat-

ed to be 70-80 g per 70 kg individual ([27] and refer-

ences cited therein). Given the molecular mass of

-glu-

tamine [147.13 AMU] and assuming 80% water content

in the body, the average concentration of

-glutamine

in human tissues is about 9 mM. On the other hand,

the concentration of kynurenine in mammalian tissues

is much lower. The concentration of kynurenine is re-

ported to be 20nmol/g (~22μM) in rat liver, with much

lower concentrations in the brain, lung, and spleen

[28]. Thus, the transamination of

-glutamine catalyzed

by GTK/KAT1 and GTL/KAT3 in vivo in human/mamma-

lian tissues is quantitatively likely to be orders of mag-

nitude greater than that of

L-kynurenine[13].

In addition to catalyzing transamination reactions,

both GTK and GTL catalyze competing β-elimination

reactions with many cysteine S-conjugates [Eq. (10)],

where -SR is a good leaving group [e.g., [13, 21, 29-32].

Because GTK was the first enzyme recognized to have

strong cysteine S-conjugate β-lyase activity ([32] and

references cited therein) the alternative abbreviation

CCBL1 (cysteine S-conjugate beta-lyase 1) is given in

UniProt for GTK/KAT1. This name is also used by some

commercial vendors. Inasmuch as GTL/KAT3 also has

strong cysteine S-conjugate β-lyase activity [30]. This

enzyme has been given the alternative name CCBL2 in

the UniProt and by some commercial vendors.

In addition, Commandeur et al. [29] have shown

that selenocysteine Se-conjugates are excellent trans-

aminase and β-lyase substrates of highly purified rat

liver GTK [Eq.(11)].

RSCH

CH(CO

–

)(NH

) + H

O ⇆

C(O)CO

–

+ RSH

(10)

RSeCH

CH(CO

–

)(NH

) + H

O ⇆

C(O)CO

–

+ RSeH

(11)

Substrate specificity of GTK and GTL. The wide

L-amino acid and α-keto acid specificity noted by Coo-

per and Meister for rat kidney GTK and rat liver GTL

[14, 15] was also noted for human KAT1 (GTK) [19] and

mouse KAT3 (GTL) [20]. In general, amino acid sub-

strates have the general structure RCH(CO

–

)NH

and

α-keto acid substrates have the structure RC(O)(CO

–

)

where R is a relatively hydrophobic, non-charged

group. Thus, for example, methionine and leucine (and

their respective α-keto acids) in addition to glutamine,

are good substrates of both enzymes [14, 15]. Valine

and isoleucine and their corresponding α-keto acids,

however, are poor substrates [14, 15, 19, 20], presum-

ably due to steric hindrance at the active site as a re-

sult of branching at the β position. Interestingly, gly-

oxylate [HC(O)(CO

–

)] (but not glycine [CH

(CO

–

)NH

])

is a good substrate of both GTK and GTL, presumably

because its small size allows ready entry at the ac-

tivesite.

METABOLIC IMPORTANCE

OF THE GTωA PATHWAY

Here we summarize the suggested metabolic role

of this pathway as recently reviewed in [13].

Closure of the methionine salvage pathway

[Eq. (12)]. Strong evidence suggests that the GTωA

pathway is largely responsible for closure of the methi-

onine salvage pathway in mammals ([13] and referenc-

es cited therein). Moreover, glutamine transaminase

and ω-amidase have been shown to act in tandem to

close the methionine salvage pathway in plants and

bacteria [33].

L-Glutamine + α-keto-γ-methiolbutyrate (KMB) +

O → α-ketoglutarate + L-methionine +

(12)

Salvage of α-keto acids. We have proposed that

the glutamine transaminases may act as repair enzymes

COOPER, DENTON1664

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

to correct the “mistakes” of other transaminases. Such

mistakes may generate α-keto acids that are potential-

ly toxic/neurotoxic at elevated levels (e.g., phenylpy-

ruvate, p-hydroxyphenylpyruvate). Owing to the irre-

versibility of the GTωA pathway, the α-keto acid will be

favorably converted back to the less toxic amino acid,

while at the same time providing anaplerotic α-keto-

glutarate ([13] and references cited therein). Caligiore

etal. suggest that GTK may be especially important in

salvaging tryptophan from its corresponding α-keto

acid [34].

Detoxification of α-keto acids. Excess glyoxylate

may be toxic due to its potential conversion to oxalate.

Highly insoluble calcium oxalate is a major contributor

to kidney stones. It has been suggested that glutamine

transaminases/KATs may divert glyoxylate to glycine

in the kidney thereby lowering the possibility of the

conversion of glyoxylate to oxalate [35].

Possible antioxidant role of the GTωA pathway.

Many α-keto acids are oxidatively decarboxylated by

which, in the process, is converted to H

O  ([36]

and references quoted therein). For example, α-ke-

toglutarate is oxidized by H

to succinate and CO

[Eq.  (13)]. We have suggested that the GTωA path-

way, as a source of α-ketoglutarate, may play a role

in antioxidant defenses and upregulation of defense

systems under hypoxic conditions [13]. Moreover, it

has been recently shown that oxidation of specific

cysteine residues in ωA by H

inhibit ωA’s catalytic

activity which, in turn, would decrease the produc-

tion of the α-ketoglutarate, making it less available to

neutralize H

[37]. In light of these new findings,

although α-ketoglutarate itself does react with H

it now seems less likely that the α-ketoglutarate,

which has come directly from the GTωA pathway,

would be the α-ketoglutarate acting as an intracellular

antioxidant.

–

CCH

C(O)CO

–

+ H

→

–

CCH

–

+ CO

(13)

Possible role of the GTωA pathway in the trans-

port of α-keto acids/-amino acids. A large number

of cellular and subcellular transporters have been de-

scribed for L-amino acids and α-keto acids. We have

suggested that the GTωA pathway, coupled to an α-ke-

toglutarate-linked aminotransferase, may assist in the

transport of

L-amino acids [13]. For example, consider

transamination of an

-amino acid with α-ketoglutarate

to the corresponding α-keto acid in compartment 1

and uptake of that α-keto acid into compartment 2.

TheGTωA pathway will ensure stoichiometric appear-

ance of the corresponding L-amino acid in compart-

ment 2 [Eqs. (14), (15)]. Removal of the α-keto acid in

compartment 2 via the GTωA pathway will ensure uni-

directional movement of the α-keto acid from compart-

ment 1 into compartment 2. The compartments may be

at the cellular or subcellular level.

Compartment 1:

L-Amino acid +

α-ketoglutarate ⇆ α-keto acid +

L-glutamate

(14)

Compartment 2:

L-Glutamine + α-keto acid +

O → α-ketoglutarate + L-amino acid +

(15)

SUBSTRATE SPECIFICITY, DISTRIBUTION

AND STRUCTURE OF ω-AMIDASE

Substrate specificity. Meister and colleagues

showed that partially purified rat liver ω-amidase hy-

drolyzes the ω-monoamides of 4- and 5-C dicarboxyl-

ates such as KGM, α-ketosuccinamate (KSM; the α-keto

acid analogue of asparagine), glutaramate, succinamate

[9, 10]. However, neither glutamine nor asparagine was

found to be a substrate of this preparation of ω-ami-

dase [9, 10]. Thus, the -C(O)- in the α position may be

replaced by a -CH

- but not by a -CH(NH

)-. Subse-

quently, Hersh [16, 17] showed that rat liver ω-amidase

catalyzes hydroxaminolysis reactions with a number of

carboxamide substrates, in which water is replaced by

hydroxylamine as the attacking nucleophile, generating

the corresponding hydroxamate [Eq.  (16)]. In addition,

rat liver ω-amidase was shown to catalyze the hydro-

lysis of the terminal carboxylate esters of a number

of 5C- and 4C-dicarboxylates and some transamidation

reactions at the ω-carboxamide [16, 17]. For a detailed

discussion of these reactions see [38].

RC(O)NH

+ NH

OH → RC(O)NHOH +

(16)

Assay procedures. Meister showed that partially

purified rat liver ω-amidase exhibits a relatively sharp

pH optimum at ~9.0 for KGM, yet a much broader pH

optimum (~5.0-9.0) with KSM as substrate, despite the

apparent close similarity in structure between these

two α-keto acids [11]. An explanation was provided by

Hersh using a continuous coupled enzyme assay pro-

cedure in which the formation of α-ketoglutarate from

KGM, catalyzed by ω-amidase [Eq. (8)], is reductively

aminated by the action of GDH in the presence of am-

monia and NADH– the reverse direction of Eq.(5) [13].

The disappearance of NADH is continuously monitored

spectrophotometrically at 340 nm (ε = 6220 M

–1

Hersh showed that, at the enzyme concentration used

in the assay, the rate of change of NAD

production at

pH values below 8.0 is biphasic [16]. From this, Hersh

was able to calculate the ratio of open-chain form

(0.3%; substrate) to the lactam form (99.7%; non sub-

strate). [Nevertheless, throughout this review, unless

stated otherwise, it is understood that the term KGM

refers to the sum of open-chain form plus lactam.]

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1665

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Hersh concluded that the rate of interconversion

between open-chain and cyclized forms of KGM is

–

OH-catalyzed. As a result, assays of ω-amidase with

KGM as substrate are typically carried out in buffers at

pH8.5-9-0 where rate of ring opening is unlikely to be

rate limiting for amounts of enzyme used in atypical

assay [39].

An alternative assay procedure uses succinamate

[R =

–

- in Eq. (16)] as substrate. ω-Amidase

catalyzes the conversion of succinamate to the cor-

responding hydroxamate in the presence of hydrox-

ylamine. The hydroxamate exhibits a brown color

(ε=920M

–1

at 535nm) when acidic ferric chloride

is added to the reaction mixture  [39]. The advantages

of this assay are that succinamate, unlike KGM, is

commercially available and the assay can be carried

out at physiological pH values (i.e., ~7.0-7.4). A third

assay procedure involves the use of KGM as a sub-

strate dissolved in buffers at pH8.5-9.0. The reaction is

quenched by addition of acidic 5mM 2,4-dinitrophenyl-

hydrazine. After incubation for a few minutes, the pH

of the reaction is adjusted by the addition of 1  M KOH

and the absorbance of the 2,4-dinitrophenylhydrazone

is measured at 430 nm (ε ~ 16,000 M

–1

) [39].

Distribution of ω-amidase. The enzyme activ-

ity is ubiquitously expressed in nature, attesting to

its central importance in nitrogen metabolism in the

biosphere. For example, Meister showed that ω-ami-

dase activity (KGM and KSM as substrates) is present

in all eight rat tissues investigated [11]. It was also

shown to be present in a Novikoff tumor, lettuce leaves,

spinach leaves, Escherichia coli and Streptococcus fae-

calis [11]. In another study, ω-amidase activity was

shown to be present in all ten rat organs investigated,

with highest specific activity in liver and kidney [40].

Prostate was not included in these studies, but lat-

er work showed that glutamine transaminase and

ω-amidase specific activities are exceptionally high in

that organ [41]. Lin et al noted the presence of Nit2

(nitrilase-like protein  2) protein in all sixteen human

tissues/cells investigated with highest levels in liver

and kidney [42]. [Nit2 is identical to ω-amidase (see be-

low)]. GeneCards indicates that the message for ω-ami-

dase/Nit2 is present in all 37 human tissues investi-

gated [43].

ω-Amidase is well represented in Neuropora cras-

sa where it is thought to be an important intermediate

in the cycling of glutamine nitrogen [44, 45]. In plants,

transamination of asparagine to KSM with glyoxylate

as co-substrate [Eq. (17)] followed by hydrolysis of

KSM, catalyzed by ω-amidase [Eq.(18)], is thought to

play a key role in photorespiration– the net reaction

is shown in Eq.(19) [46, 47].

L-Asparagine + glyoxylate →

α-ketosuccinamate (KSM) + glycine

(17)

KSM + H

O → oxaloacetate +

(18)

Net:

L-Asparagine + glyoxylate + H

O →

oxaloacetate + glycine +

(19)

Special role of an ω-amidase in microbial nico-

tine metabolism. Gram-positive soil bacteria Arthrobac-

ter nicotinovorans, Nocardioides sp. JS614 and Rhodo-

coccus opacus were shown to contain a similarly

organized gene cluster for the catabolism of nicotine

[48]. An intermediate in the catabolism of nicotine by

A. nicotinovorans was shown to be KGM and one of

the genes in the gene cluster was found to code for

an enzyme that hydrolyzes KGM to α-ketoglutarate and

ammonia [48]. The enzyme was found to have a mono-

mer mass of 32.3  kDa – only slightly larger than that

of the mammalian ω-amidase monomer (30.6  kDa; see

below)– and possess a characteristic EKC triad at the

active site [48]. Interestingly, the mammalian ω-ami-

dase is a homodimer (see below), whereas preliminary

data suggested that the A.  nicotinovorans enzyme is a

homotetramer [48].

Structure and location of mammalian ω-ami-

dase/Nit2. The enzyme is annotated as Nit2 in human

and mammalian gene data banks – see [49, 50]. The

human enzyme (Q9NQR4 (UniProt) [51]) is a homodi-

mer; each monomer consists of 276 amino acids with

a molecular mass of 30,608Da. Predicted three dimen-

sional structures for the human enzyme are report-

ed in [52] and [53] – the AlphaFold predicted struc-

ture [AF-Q9NQR4] is classified as “very high, with a

pLDDT>90. The enzyme active site on each monomer

possesses a reactive cysteine residue [16, 17], part of a

glutamate, lysine, cysteine triad (E43; K112; C153 in the

human enzyme) [52, 53].

Cellular and extracellular location of ω-Ami-

dase/Nit2. The enzyme is present in rat liver cytosol

[10,  40] and mitochondria [40,  54]. The mechanism

for the import of ω-amidase into mitochondria re-

quires a detailed study. Interestingly, UniProt lists a

protein– epididymis secretory sperm binding protein

Li  8a (Q9NQR4-1) that has a sequence identical to that

of ω-amidase and is present in the centrosome [51].

However, we are unable to find any additional informa-

tion on the apparent identity of ω-amidase/Nit2 with

this binding protein. Finally, it has been shown that

Nit2 (i.e., ω-amidase) is one of 1160 proteins identified

in urinary exosomes by the NHLBI Epithelial Systems

Biology Laboratory [55].

Possible post translational modification sites.

PhosphSitePlus® indicates about thirty potential sites

for post translational modifications of Nit2/ω-amidase

of which Y49 (phosphorylation), Y145 (phosphorylation)

and K249 (acetylation) are the most prominent [56].

In this context, it was previously noted that folate

deficiency in human colonocytes in culture resulted

COOPER, DENTON1666

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

in a remarkable, ~98% loss of Nit2 (i.e., ω-amidase)

protein, compared to that of control cultures as as-

sessed by staining intensity of the protein spot on a

2D gel [57]. Possibly, Nit2 is phosphorylated under low

folate conditions and moves to a different position on

the 2D gel. Moreover, in a proteomic study of MCF7

breast cancer cells, a shift in isoelectric point of Nit2

(i.e., ω-amidase) compared to normal breast cells was

noted, but only in those cancer cells overexpressing

ERBB2 (Erb-B2 receptor tyrosine kinase) [58]. Whether

phosphorylation of the enzyme under normal physi-

ological conditions occurs in vivo, and whether this

plays a role in regulating enzyme activity and location

requires a detailed study.

ω-AMIDASE/Nit2 IS A MEMBER

OF THE NITRILASE SUPERFAMILLY

The nitrilase superfamily of enzymes, which in-

cludes nitrilases, amidases and amidotransferases, has

been classified into 13 families [59,  60]. ω-Amidase/

Nit2 [along with Nit  1 (deaminated glutathione (dGSH)

amidase [61])] are the sole mammalian members of

family 10 of the nitrilase superfamily. All members of

the superfamily, including ω-amidase, contain a canon-

ical glutamate  (E), lysine  (K), cysteine  (C) triad at the

active site [60, 62]. Interestingly, this superfamily does

not include the two mammalian glutaminase isozymes.

The architecture of the active site in the two mamma-

lian glutaminase isozymes is different from those of

the nitrilase superfamily and the active site contains

a reactive serine residue rather than a reactive cys-

teine residue – see below. The importance of the EKC

triad (i.e., E43, K112, C153) for the catalytic activity in

human ω-amidase is shown by mutation studies. Sepa-

rate mutations of each of these residues to an alanine

residue results in an inactive enzyme  [52]. Moreover,

removal of the loop that forms a lid over the active

site (residues 116-128) also results in loss of activity

[52]. In addition to the EKC triad, according to Weber

et al. “All known nitrilase superfamily amidase and

carbamoylase structures have an additional glutamate

that is hydrogen bonded to the catalytic lysine” [62].

Geobacillus pallidus amidase catalyzes the hydrolysis

of simple aliphatic amides [62]. Both an E(142)L mu-

tation and an E(142)D mutation result in an inactive

enzyme [62]; see also [63]. Interestingly, one of the

substrates studied in the latter study was glutaramate,

which is also a substrate of ω-amidase [11, 17].

Yeast Nit2 (yNit2) is an ortholog of mamma-

lian Nit1 but NOT of mammalian Nit2. The crystal

structure of yeast Nit2 (yNit2) has been reported by

Liu et al. [64]. Confusingly, yNit2 is more related to

mammalian Nit1 than to mammalian Nit2 ([61, 64] and

references cited therein). A compound that is present

in the active site of yNit2 was tentatively identified as

GSH-like by Liu etal. [64]. Subsequently, Peracchi etal.

[61] showed that dGSH is a major substrate of both

yNit2 and mouse Nit1 (mNit1) [61]. KGM is a relatively

poor substrate of both yNit2 [61, 64] and mNit1 [64].

As noted by Perrachi etal. for mouse Nit2 (mNit2)[61]

“Sequence comparisons and structural data indicate

that the residues of the Nit2 catalytic pocket that sur-

round the substrate α-KGM are conserved in Nit1.”

A major difference is, however, found in the subsite

where the amido group of α-KGM is predicted to bind,

which is a quite small niche in the structure of Nit2,

but a much larger cavity in Nit1 – see also [65,  66].

These considerations are elaborated further in the

next section which discusses the mechanism of human

ω-amidase/Nit2.

Catalytic mechanism of ω-amidase/Nit2. Based

on a QM/MM study of hNit2 (i.e., human ω-amidase)

a theoretical four-step process that includes two tran-

sition states (TSs) at the active site during turnover

of KSM to oxaloacetate and ammonia has been sug-

gested [53]. This mechanism, depicted using KGM as

the substrate and the numbering of the human EKC

triad (i.e., Glu

, Lys

112

, Cys

153

), is depicted in Fig.  2.

In the first step, the amide group of the substrate is

activated by coordination with Lys

112

of the catalytic

triad, and the supporting Glu

128

residue, followed by

nucleophilic attack by Cys

153

. The stabilized tetrahedral

intermediate collapses to form the thioester interme-

diate with concomitant loss of ammonia/ammonium.

Subsequently, a water molecule, activated by coor-

dination to Glu

, attacks the thioester intermediate

releasing KG followed by redistribution of active site

residues for a subsequent round of catalysis. Curiously,

the authors mention that the enzyme has 276 resi-

dues (as also noted above for human ω-amidase and

in UniProt [51]) yet show the active site residues to be

Glu81, Lys150, and Cys191, which is shifted by 38 ami-

no acids. We assume that the authors recapitulate the

residues mentioned in the work of Barglowetal. [66]

and also assume that the 38 additional residues are a

product of the expression system used for production

of the enzyme (N-terminal hexahistidine fusion with

the gene 10 leader sequence).

ω-AMIDASE/Nit2 – CLINICAL ASPECTS

Differentiation of Crohn’s disease (CD) from

ulcerative colitis (UC). Burakoff et al. investigated

whether blood-based biomarkers can differentiate UC

from CD and noninflammatory diarrhea [67]. These

authors generated whole blood gene expression pro-

files for 21 patients with UC, 24 patients with CD, and

10 control patients with diarrhea, but without colon-

ic pathology. According to the authors: “A supervised

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1667

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Fig. 2. Proposed reaction mechanism for the hydrolysis of KGM by human ω-amidase. The catalytic triad residue numbers

reflect that on the human enzyme correlating to that of Chienetal. [52]. Molecular modeling of the mechanism was performed

by Silva Teixeiraetal. [53] and are the basis of the mechanism proposed.

learning method (logistic regression) was used to iden-

tify specific panels of probe sets which were able to

discriminate between UC and CD and from controls.

The UC panel consisted of the four genes, CD300A,

KPNA4, IL1R2, and ELAVL1; the CD panel comprised

the four genes CAP1, BID, NIT2, and NPL. These panels

clearly differentiated between CD and UC” [67]. Note

the inclusion of Nit2 (i.e., ω-amidase) in the CD panel.

ω-Amidase/Nit2 as a possible diagnostic biomarker in

CD requires further study.

Nit2 and Down syndrome. Myung et al. carried

out MALDI-TOF mass-spectrometric identification and

quantification of spots on 2D gel electrophoresis after

in-gel digestion of proteins using cortical brain sam-

ples from 7 controls and 7 samples from fetal Down

syndrome at the early second trimester  [68]. Three

proteins tentatively identified as reduced by 50% in

Down syndrome fetal brain versus normal fetal brain

were identified as Rik protein, mitochondrial inner

membrane protein and Nit2 [68]. The significance of

this finding also requires further study.

Role of ω-amidase/Nit2 in tumor progression.

Nit1, as noted above, is now known to be a dGSH de-

aminase. The enzyme catalyzes the hydrolysis of dGSH

to α-ketoglutarate and cysteinylglycine [61]. Consider-

able evidence suggests that Nit1 is a tumor suppres-

sor (e.g., [69-71]). Because Nit2 has moderate sequence

similarity to Nit1, Lin et al. considered the possibili-

ty that Nit2 (i.e., ω-amidase) is also a tumor suppres-

sor [42]. Ectopic expression of Nit2 in HeLa cells was

found to inhibit cell growth through G(2) arrest rather

than by apoptosis [42]. The authors also showed, using

proteomic and RT-PCR analysis, that Nit2 up-regulated

the protein and mRNA levels of 14-3-3sigma, an inhib-

itor of both G(2)/M progression and protein kinase B

(Akt)-activated cell growth, and down-regulated 14-3-

3beta, a potential oncogenic protein [42]. The authors

concluded that Nit2 may be a tumor suppressor can-

didate [42]. However, other workers have concluded

that Nit2 may be a cancer promoter in colorectal can-

cer [72]. Moreover, the authors suggested that Nit2

may be a promising target for the treatment of colon

cancer [72]. In another study, it was shown that the

expression of Nit2 in tongue squamous cell carcino-

ma is significantly higher than that in normal tongue

tissues [73]. The authors also suggested that Nit2 may

be a therapeutic target [73]. As noted above, GTK and

ω-amidase are well represented in rat prostate tissue

[41]. Furthermore, it was shown that protein levels

of these enzymes increased in human prostate cells

in culture in tandem with the aggressiveness of the

cancer [41]. We suggest that, on balance, ω-amidase

COOPER, DENTON1668

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

is a tumor promoter by providing anaplerotic α-ketoglu-

tarate to rapidly dividing cancer cells (see the next sec-

tion).

Glutamine addiction in tumors – the canonical

pathway. It is well known that many cancers utilize

glutamine as a major source of anaplerotic α-ketoglu-

tarate and nitrogen for DNA synthesis. This has been

termed “glutamine addiction” ([13] and references cited

therein). Most researchers in the field only consider one

pathway for the conversion of glutamine to α-ketoglu-

tarate. This pathway (the canonical pathway) consists

of the conversion of glutamine to glutamate via the

action of a glutaminase, followed by conversion of glu-

tamate to α-ketoglutarate by a transamination reaction

[Eqs. (20)-(22)]. Note that Eq.(21) depicts the reverse

direction of Eq.(4). Alternatively, glutamate may be con-

verted to α-ketoglutarate by the action of GDH [Eq.(5)].

L-Glutamine + H

O → L-glutamate +

(20)

L-Glutamate + α-keto acid ⇆

α-ketoglutarate +

L-amino acid (21)

Net:

L-Glutamine + H

O + α-keto acid →

α-ketoglutarate +

L-amino acid +

(22)

Human cells contain two glutaminase isozymes

encoded by GLS and GLS2. GLS encodes a kidney-type

glutaminase (KGA) – also known as GLS1 (or simply

GLS). GLS also encodes its active, shorter splice variant

(glutaminase C, GAC). GAC is present in many cancers

([74] and references quoted therein) and is thought

to be a major enzyme contributing to their glutamine

addiction. GLS2 encodes an enzyme (GLS2) that is

normally predominant in the liver and also exists as

splice variants– LGA and GAB (glutaminaseB) [75-77].

In addition, LGA exists as two isoforms, one of which

is more active than the other [76]. There appears to be

some confusion in the literature on the naming of the

more active isoform of GLS2 [75-77]. In their recent

article, Feng et al., use the term LGA to describe the

more active GLS2 isoform [74]. Unlike GLS1, which is a

tumor promoter in many cancers, GSL2 may be either

a tumor promoter or tumor suppressor [74, 78].

An Aside – Glutaminases involved in glutamine

addiction – can their catalytic properties reveal

insights into ω-amidase catalysis and regulation?

Many enzymes are activated by forming polymers

[79,  80]. Perhaps the most well studied such enzyme

is acetyl-CoA carboxylase ([80] and references cited

therein). The enzyme is active as a homodimer, but

its activity is greatly increased upon polymerization.

Inaddition, the activity is regulated in a complex fash-

ion by phosphate binding and by allosteric regulators

[80]. It is now becoming apparent that the mamma-

lian glutaminases are activated in a similar fashion.

GLS1 can exist as an inactive dimer as well as an

active tetramer [80-82]. Moreover, both glutaminase

isozymes are activated by phosphate and contain al-

losteric binding sites ([74] and references cited therein).

Inaddition, it is known that GLS1 (i.e., KGA), GAC (i.e.,

active shortened form of KGA) and GLS2 can exist

in active polymeric forms [74,  81-83]. Recently, Feng

et  al. demonstrated that the ability of GAC and GLS2

to form filaments is directly coupled to their catalytic

activity  [74]. The authors further noted that “Filament

formation guides an ‘activation loop’ to assume a spe-

cific conformation that works together with a ‘lid’ to

close over the active site and position glutamine for

nucleophilic attack by an essential serine.” The active

site of GLS1 is different from that of ω-amidase. For

example, as noted above, the active site of GLS/GAC

contains a reactive serine residue [74], whereas that of

ω-amidase contains a reactive cysteine residue [16,  52,

53]. However, a point of similarity is that both enzymes

require a lid to cover the active site for catalysis to

proceed [52,  53,  74]. Nothing is currently known about

possible regulation of ω-amidase activity. Nevertheless,

based on the fact that KGM and glutamine are almost

identical in size (differing in molecular mass by just

1  Da) and are monoamides of 5-C dicarboxylic acids,

it is possible that ω-amidase shares some regulatory

features with GLS1 and GLS2. This is another area that

requires detailed study.

Glutamine addiction in tumors – the alterna-

tive GTωA pathway for the formation of anaple-

rotic α-ketoglutarate. Several clinical studies have

been completed or are ongoing for the treatment of

various cancers with the allosteric glutaminase inhib-

itor CB-839 monotherapy or in combination therapy

with other drugs (e.g.,[84-87]). Whereas some of these

treatments seem to be well tolerated, they have only

been moderately successful. We have suggested that

this may be due, in part, to an alternative pathway for

the conversion of glutamine to α-ketoglutarate – i.e.,

the GTωA pathway (originally referred to as the gluta-

minase  II pathway), that can provide anaplerotic α-ke-

toglutarate to cancer cells when GLS1 is inhibited [13,

88]. Although this pathway has mostly been ignored

(or unrecognized) by cancer researchers, strong ev-

idence has recently been presented that the GTωA

pathway is a major source of anaplerotic α-ketogluta-

rate in pancreatic cancer. Thus, Udupa et al. showed

that genetic suppression of GTK in pancreatic tumors

(P198 shGTK-KD cancer cells injected in the back of

nude mice) led to complete suppression of pancreatic

tumorigenesis [89]. The authors suggested that a GTK

inhibitor may be useful, either alone or in combina-

tion with a GLS1 inhibitor, for the treatment of cancer

[89]. In another study, Pham et al., investigated the

isotopomer patterns in

L-glutamate generated from

L-[U-

C]glutamine, in orthotopic human Myc-amplified

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1669

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

D425MED medulloblastoma tumor and showed that

this tumor preferentially uses the GTωA pathway over

the canonical GLS1 pathway to convert L-glutamine

to L-glutamate (in this case, via α-ketoglutarate) [90].

The authors noted that KYAT1 (i.e., the gene for GTK)

and its mRNA are upregulated in medulloblastoma

compared to other pediatric brain tumors in the Chil-

dren’s Brain Tumor Network/KidsFirst Pediatric Brain

Tumor Atlas RNAseq dataset [90]. In another study, as

noted above, not only does GLS1 protein increase in

prostate cancer cells in culture as the aggressiveness

of the tumor derived therefrom increases, but GTK

and ω-amidase proteins also increase concomitantly

[41]. It has been suggested that inhibitors of gluta-

mine transaminases may be useful anti-cancer agents,

perhaps in combination with GLS1 inhibitors [41, 89,

90]. Glutamine transaminase inhibitors are discussed

in the next section.

Glutamine transaminase inhibitors. Transition

state (TS) mimetics that inhibit KMB transamination

in cells in culture have been developed, including

L-methionine ethyl ester pyridoxal (MEEP) [91, 92].

Inasmuch as KMB is a substrate of both GTL and GTK,

it is reasonable to assume that these TS mimetic inhib-

itors will block L-glutamine transaminases. Interesting-

ly, MEEP was shown to induce DNA strand breaks in

HeLa cells typical of apoptotic cell death [92]. Some

inhibitors of KAT1 (i.e., GTK) have been synthesized

with K

values ranging from ~20 µM to ~1 mM [93] but,

to our knowledge, have not been tested as anti-can-

cer agents. The anti-cancer agent cisplatin is known

to be nephrotoxic at least in part due to formation of

the corresponding cysteine S-conjugate [94]. Mitochon-

drial AspAT is known to catalyze cysteine S-conjugate

β-lyase reactions [95]. Thus, it was reasoned that this

enzyme might catalyze a β-lyase reaction with the

cysteine S-conjugate of cisplatin. Overexpression of

mitAspAT in LLC-PK

cells led to increased toxicity of

cisplatin, increased platination in mitochondria and

increased inhibition of α-ketoglutarate dehydrogenase

complex, possibly due to close juxtapositioning of mi-

tAspAT to the enzyme complex [96].

In that study (i.e., [96]), GTK was not studied as

a possible β-lyase contributing to the nephrotoxicity.

Nevertheless, in a recent study, Sukeda etal. purified

human recombinant CCBL1 (i.e., GTK/KAT1) and used

a high-throughput screening assay (transamination be-

tween kynurenine and pyruvate) to screen chemical

libraries (not specified) for potential inhibitors [97].

2′,4′,6′-Trihydroxyacetophenone (THA; a naturally oc-

curring metabolite in Curcuma comosa) was identified

as a dose-dependent inhibitor of the human recombi-

nant GTK/KAT1 with an IC

of 13.2 μM [97]. The au-

thors stated that THA inhibited the β-lyase activity,

but inspection of their data shows that the inhibition

was actually of the transaminase activity [97]. Never-

theless, the authors noted some protective effects of

THA against cisplatin-induced toxicity toward mouse

kidney in vivo and LL-C-PK1 cells in culture. However,

the compound did not prevent the proliferation of can-

cer cell lines– LLC and MDA-MB-231 [97]. Sukeda etal.

also recognized other potential caveats. For example,

THA may have been protective by acting as an antiox-

idant. Nevertheless, THA could be the lead compound

in the search for compounds that will result in more

powerful glutamine transaminase inhibitors.

In conclusion, much work remains to be carried

out in the search for a selective and potent inhibitor

of GTK (and GTL). Such an inhibitor will be useful in

studies of the enzyme mechanism, but it may have

limitations as an anti-cancer agent due to the possi-

ble involvement of the glutamine transaminases in so

many physiologically relevant processes (see the above

discussion). For this reason, compounds designed to

disrupt the GTωA pathway may be better directed to-

ward inhibition of ω-amidase rather than toward in-

hibition of glutamine transaminases. We will return to

this point later.

ENDOGENOUS CONCENTRATIONS

OF α-KETOGLUTARAMATE (KGM) IN NORMAL

RAT TISSUES AND HUMAN BODY FLUIDS

Despite the fact that ω-amidase is inherently of

high specific activity in tissues, it is now well estab-

lished that KGM is an endogenous, natural metabolite.

This is presumably due to the cyclization of open-chain

(substrate) form to a lactam (inactive as a substrate),

and that the rate of ring opening is relatively slow at

neutral pH [16]. Because the rate of ring opening is hy-

droxide ion (

–

OH)-dependent, any pathological condi-

tion that will result in lowering of the cellular pH (e.g.,

hypercarbia [98]) will impede the ω-amidase reaction

with KGM even further.

The occurrence of KGM as a natural metabolite

was first observed in human cerebrospinal fluid (CSF).

In the procedure used by the investigators, endogenous

KGM was converted to α-ketoglutarate with ω-amidase

purified from rat liver. The generated α-ketoglutarate

was then measured fluorometrically with GDH in the

presence of NADH and ammonia [99-101]. The pres-

ence of KGM in human CSF was later verified by us-

ing a gas chromatography-mass spectrometry (GC-MS)

procedure [102]. KGM has also been measured in hu-

man urine by a GC-MS procedure [103, 104]– see also

below. Both GC-MS procedures measure the cyclized

lactam form of KGM. In another procedure, KGM was

measured in human CSF rat liver, rat kidney and rat

brain by an isotope dilution method [105]. Finally, a

high-performance liquid chromatography (HPLC) pro-

cedure has been developed to measure underivatized

COOPER, DENTON1670

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

KGM in rat tissues and plasma [106]. Taken together,

the data indicate that the concentration of KGM in nor-

mal human CSF is ~5 μM, whereas the concentration

in rat liver, kidney, brain, and plasma is ~8-216, 5-13,

6-11, and 19 μM, respectively [99, 101, 105, 106]. The

concentration in normal human urine is ~1-3 μmol

KGM/mmol creatinine [103]. Halámková and colleagues

showed that two separate spectrophotometric coupled

enzyme assays could be used to determine the con-

centration of KGM estimated to be present in human

serum [107]. The authors estimated that the concen-

tration of KGM in normal human plasma is ~5-10 μM

[107]– a concentration in the same order of magnitude

as that noted by Shurubor etal. of 19μM [103].

CLINICAL STUDIES IN WHICH KGM

WAS MEASURED/DETECTED IN BODY FLUIDS

Increased concentrations of KGM in the CSF

of patients with liver disease. Duffy and colleagues

showed that KGM occurs in human CSF and that the

concentration is increased in hyperammonemic pa-

tients with hepatic encephalopathy (HE) [99, 101, 105].

Moreover, the concentration was found to increase in

proportion to the severity of the disease. In some se-

vere cases of HE, CSF KGM concentrations exceeded

50  μM [99,  105]. A strong correlation was also noted

between concentrations of KGM and glutamine in the

CSF [99,  105]. Most likely, increased ammonia in the

HE patients resulted in increased tissue glutamine

concentrations, which in turn contributed to increased

transamination of glutamine. Mild neurotoxic effects

were noted upon infusion of KGM into rat CSF [99,

105]. This finding led Duffy etal. to suggest that KGM

may be a neurotoxic agent contributing to the enceph-

alopathy noted in HE patients [99,  101,  105]. However,

the concentration of KGM in the infusate (10  mM) is

>2000-fold greater than that expected in normal CSF

and 100-fold greater than the highest concentration

found in the CSF of an HE patient.

KGM exhibits a red color when spotted onto fil-

ter paper (or onto paper chromatograms) and then

sprayed with an ethanolic solution of Ehrlich’s reagent

[(4-dimethylamino)benzaldehyde] [108]. The procedure

is sensitive enough to detect KGM at a concentration

of 20  μM in 20  μL of CSF, spotted directly onto paper

without prior treatment [108]. The chemistry behind

this reaction is unknown, but the spot test may pos-

sibly serve as a starting point for the development of

a clinical test for KGM.

The KGM used in the studies of Duffy et al. was

synthesized by the method of Meister [11] in which

a solution of glutamine is incubated with snake ven-

L-amino acid oxidase in the presence of catalase,

followed by purification of KGM by cation exchange

chromatography. However, it has been known for more

than 75 years that glutamine spontaneously cyclizes

in solution to 5-oxoproline (5-OP) and formation of

ammonia [109]. Thus, even the best preparations of

KGM prepared by this enzymatic procedure contain a

few percent 5-OP. This compound is known to be en-

zymatically converted to

L-glutamate [110] – an amino

acid in the CSF suggested to be an excitotoxin even

at μM concentrations (e.g., [111,  112] and references

cited therein).

Increased urinary KGM/creatinine ratios in

primary and secondary hyperammonemia. Prima-

ry hyperammonemia is due to a defect that directly

affects enzymes or transporters of the urea cycle [113].

Secondary hyperammonemia occurs when the func-

tion of the urea cycle is inhibited by toxic metabolites

or by substrate deficiencies [113, 114]. The possibility

that increased production of KGM also occurs in pa-

tients who do not have overt liver disease but have

an inborn error of metabolism resulting in primary

hyperammonemia was investigated by Kuhara and col-

leagues [103]. The KGM/creatinine ratio was shown to

be markedly elevated in urine obtained from patients

with primary hyperammonemia due to an inherited

metabolic defect in any one of the five enzymes of

the urea cycle [103]. Kuhara etal. also noted increased

urinary KGM in three patients with a defect result-

ing in lysinuric protein intolerance and one of two

patients with a defect in the ornithine transporter  I

[103]. It was suggested that the increase in urinary

KGM concentrations in patients with primary hyper-

ammonemia is related to concomitant increase of glu-

tamine concentration [103]. On the other hand, urinary

KGM levels were not well correlated with secondary

hyperammonemia in patients with propionic acidemia

or methylmalonic acidemia, possibly as a result, in

part, of decreased glutamine levels [103].

In another study, Kuhara and colleagues noted an

increase in urinary KGM in most patients with second-

ary hyperammonemia resulting from citrin deficiency,

despite normal levels of urinary glutamine [104]. Citrin

is a hepatic mitochondrial aspartate-glutamate carrier

coded by the SLC25A13 gene and a defect in this gene

can lead to episodic hyperammonemia and disturbed

consciousness ([115] and references cited therein).

Frainay et al. have devised a network-based rec-

ommendation system to interpret and enrich metab-

olomics results that they have named MetaboRank

[116]. Interestingly, the authors state “...MetaboRank

recommended the overlooked α-ketoglutaramate as a

metabolite which should be added to the metabolic

fingerprint of HE…” [116].

In summary, KGM may be a useful biomarker for

many hyperammonemic diseases including hepatic en-

cephalopathy, inborn errors of the urea cycle, citrin de-

ficiency and lysinuric protein intolerance.

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1671

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Strong association of urinary KGM with uro-

modulin in chronic kidney disease patients. In a

recent study, it was shown that KGM is one of five me-

tabolites (out of 607) that strongly (P 2.11e

–440

) correlat-

ed with the protein uromodulin (UMOD) in a cohort

of kidney disease patients (n  =  462) in Germany [117].

The kidney-specific UMOD has been suggested to be a

potential marker for tubular functional mass in pop-

ulation-based studies [117-121]. According to Pruijm

et al. [118] the associations of UMOD excretion with

markers of tubular functions and kidney dimensions

may reflect tubule activity in the general population.

It has also been suggested to be a marker for structur-

al integrity of the distal nephron, and renal function

[120, 121]. The reason for the association of KGM with

uromodulin is not obvious and requires further study.

KGM as a potential biomarker in serum ob-

tained from stroke victims. Sidorov etal. used me-

tabolomic analysis to prospectively analyze potential

serum biomarkers in acute ischemic (7 h) and chronic

(3-6 months) stages of 60 stroke victims [122]. The au-

thors noted the presence of four unknown metabolites

at the acute stage that were significantly associated

with infarct volume (all p < 0.01) [122]. Nine metabo-

lites at the chronic stage were found to significantly

associate with infarct volume, namely 3-indolepropio-

nate, α-ketoglutaramate, picolinate, and six unknowns

(all p<0.048) [122].

OTHER STUDIES INVOLVING

BIOLOGICALLY OCCURING KGM

Plasma and tissue KGM in rats treated with the

hepatotoxin thioacetamide. Shurobor et al. used an

HPLC method to show that the concentration of KGM

in normal rat plasma is ~19  μM [106,  123]. The au-

thors treated rats with various doses (200, 400, and

600mg/kg) of hepatotoxic thioacetamide and, after six

days of recovery, measured the concentration of sever-

al TCA cycle metabolites, and that of KGM, in the plas-

ma [123]. Despite the apparent physiological recovery

of the rats at this time, a metabolic imbalance was still

apparent [123]. Notably, the concentration of plasma

KGM was decreased by about 15-20% in rats treated

with all three doses, relative to that of a control [123].

The authors noted that the concentration of KGM in

liver and kidney, but not in brain, tended to decrease

in proportion to the amount of thioacetamide adminis-

tered [123]. The α-ketoglutarate/KGM ratio was signifi-

cantly increased in brain and kidney as the concentra-

tion of administered thioacetamide increased, but the

ratios in the kidney were not significantly different

from that of the control [123].

Increased serum KGM in dogs with exocrine

pancreatis insufficiency (EPI). EPI results from in-

sufficient secretion of pancreatic digestive enzymes

([124] and references cited therein). Barko etal., used

UPLC-MS/MS to carry out untargeted serum metabolo-

mics to identify metabolic disturbances associated with

EPI [124]. Fasted serum samples were collected from

dogs with EPI (n  =  20) and healthy controls (n  =  10),

all receiving pancreatic replacement therapy [122].

759 serum metabolites were detected, of which the con-

centration of 114 varied significantly (p<0.05, q  <  0.2)

between dogs with EPI and healthy controls. Of note

was a marked increase in the concentration of serum

KGM [124]. The reason for this increase requires fur-

ther study.

ADDIONAL ASPECTS

OF ω-AMIDASE/KGM BIOLOGY

Wing and eye development in Drosophila mela-

nogaster. Knockdown of the gene CG813 in D.  mela-

nogaster, that encodes a homologue of mammalian

ω-amidase, results in severe eye- [125] and wing de-

fects [126]. It is not clear whether the defects are due

to a buildup of KGM that is specifically toxic to eye

and wing development, decreased synthesis of anaple-

rotic α-ketoglutarate that specifically affects wings and

eyes, or to an as yet unidentified moonlighting prop-

erty of ω-amidase required for correct eye and wing

development.

Role of KGM in stimulating nitrogen assimila-

tion in plants. Unkefer etal. have recently shown that

KGM (also known as 2-hydroxy-5-oxoproline) stimu-

lates nitrate uptake by plants and is a “likely signal for

ammonium assimilation” [127]. These authors suggest

that application of KGM to agricultural plants “would

potentially reduce the use of nitrate fertilizer per unit

of yield, leading to a further reduction in agriculture’s

carbon footprint” [127].

THE NEED FOR LARGE SCALE

SYNTHESIS OF KGM CHEAPLY

AND ON AN INDUSTRIAL SCALE

One of the reasons why ω-amidase has been so

little studied is that the substrate KGM is not available

commercially and, until recently, could only be made

enzymatically by oxidation of

L-glutamine with snake

venom L-amino acid oxidase in the presence of catalase

[11]. However, as noted above, such preparations are

invariably contaminated with small amounts of 5-oxop-

roline and trace amounts of α-ketoglutarate. Recently,

one of us (TTD) has devised a laboratory scale organic

synthesis of KGM that is not contaminated with 5-OP

and/or α-ketoglutarate [128]. Martinez and Unkefer have

patented another organic synthesis procedure [127].

COOPER, DENTON1672

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Fig. 3. The importance of ω-amidase as a clinical marker; the central role of KGM as a clinical marker and as a marker of

nitrogen metabolism; the GTωA pathway as a source of anaplerotic α-ketoglutarate to cancer cells.

This procedure relies on the oxidation of glutamine or

5-oxoproline with Fremy’s salt. The yield is reported to

be high [129]. The possible advantages and disadvan-

tages of the procedure are discussed in [130]. In con-

clusion, (i)  given the potentially enormous agricultural

benefit of using KGM to stimulate nitrate assimilation

and (ii)  the need to study the biological and clinical

significance of ω-amidase/KGM, it is of utmost impor-

tance that methods be devised for the cheap, large-

scale synthesis of KGM.

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1673

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

THE NEED FOR KGM LABLED

WITH HEAVY ISOTOPES

Most of the metabolomic studies mentioned above

that recognized KGM as an important intermediate

were carried out by Metabolon Inc. The company

does not currently have access to authentic KGM or

a suitable internal standard. Nevertheless, Metabolon

recognizes the KGM peak in the MS profile with a high

degree of certainty based on fragmentation pattern.

In other studies, Kuhara et al. measured KGM peak

heights relative to creatinine by using authentic KGM

but, in this case, γ-methyl KGM was used as an internal

standard. Given the growing recognition of the meta-

bolic and clinical importance of KGM, it is important

that KGM, labeled with one or more heavy isotopes, be

synthesized as an appropriate internal standard and

for quantitation.

THE NEED FOR SELECTIVE

INHIBITORS OF ω-AMIDASE

Given the many roles of the pathway in interme-

diary metabolism and especially in providing anaple-

rotic α-ketoglutarate to cancer cells, it will be very

important to develop inhibitors of the enzymes of the

GTωA pathway. As noted above, some researchers have

suggested that inhibitors of GTK may be useful anti-

cancer agents, perhaps in combination with a GLS1/

GAC inhibitor. Some inhibitors of GTK have been de-

scribed in the literature. These have not been tested

in a clinical setting but can perhaps be used as lead

compounds in the development of more potent inhib-

itors. However, as also noted, selective inhibitors of

ω-amidase may be even more useful as anticancer

agents, perhaps in combination with a GLS1/GAC in-

hibitor. Unfortunately, no selective and potent inhibi-

tor of ω-amidase is currently available, although some

strategies for the development of such inhibitors have

been suggested [130].

CONCLUSIONS

ω-Amidase is ubiquitously expressed in nature,

often in high levels, yet its biological roles remain

largely unappreciated by biomedical scientists. This

review emphasizes (i)  the central role of KGM in nitro-

gen metabolism, (ii)  ω-amidase and its substrate KGM

as clinical markers and (iii)the proposed importance of

the GTωA pathway as a source of anaplerotic α-ketoglu-

tarate to cancer cells. This is schematically shown in

Fig.  3. Finally, we emphasize the need for the devel-

opment of selective inhibitors of GTK and especially

of ω-amidase.

Abbreviations.

–

OH, Hydroxide ion; 2D, Two di-

mensional; 5-OP, 5-Oxoproline (synonym: pyrogluta-

mate); ω-Amidase,ω-Amidodicarboxylate amidohydro-

lase; AlaAT,alanine aminotransferase; AspAT,aspartate

aminotransferase; BID, BH3 interacting domain death

agonist is a member of the BCL-2 family; CAP1,adeny-

lyl cyclase-associated protein-1; CCBL1,cysteine S-con-

jugate β-lyase1; CCBL2,cysteine S-conjugate β-lyase2;

CD, Crohn disease; CD300A, Cluster of differentiation

300A; CSF,cerebrospinal fluid; dGSH, deaminated glu-

tathione; EKC, Glutamate (E), lysine (K), cysteine (C);

ELAVL1,Embryonic lethal abnormal vision [synonym:

or HuR (Drosophila-like Hu antigen R)]; EPI,exocrine

pancreatic insufficiency; GABA, γ-aminobutyric acid;

GAB,glutaminaseB; GAC,glutaminaseC; dGSH,deam-

inated glutathione; GC-MS, Gas chromatography-mass

spectrometry; GDH, glutamate dehydrogenase; GLS

or GLS1, glutaminase isozyme 1 (synonyms: kid-

ney type glutaminase); GLS2, glutaminase isozyme 2

(synonyms: liver type glutaminase); GTK, glutamine

transaminase K [synonyms: kynurenine aminotrans-

ferase 1; GTL, glutamine transaminase L [synonyms:

kynurenine aminotransferase 3; GTωA, glutamine

transaminase ω-amidase; GOT,glutamate oxaloacetate

transaminase; GPT, glutamate pyruvate transaminase;

GSH, glutathione; HE, hepatic encephalopathy; HPLC,

high performance liquid chromatography; IL1R2,

interleukin 1 receptor, type  II; KAT/KYAT, kynure-

nine aminotransferase; KGA, kidney-type glutaminase;

KGM, α-Ketoglutaramate (synonyms: 2-oxoglutara-

mate, 5-amino-2,5 dioxopentanoate); KMB, α-Keto-γ-

methiolbutyrate (synonyms: 4-methylthio-2-oxobu-

tanoate, 4-methylthio2-oxobutyrate); KOH, potassium

hydroxide; KPNA4, karyo pherin alpha 4 (synonym:

importin alpha 3); KSM, α-ketosuccinamate (syn-

onyms: 2-oxosuccinamate, 4-amino-2,4-oxobutanoate);

LGA, liver type glutaminase; MALDI-TOF, matrix as-

sisted laser desorption ionization-time of flight; MEEP,

L-methionine ethyl ester pyridoxal; NADH, nicoti-

namide adenine dinucleotide; mNit2, mouse nitri-

lase like protein 2; NPL, N-Acetylneuraminate pyru-

vate lyase; mRNA, messenger ribonucleic acid; Nit2,

Nitrilase like protein 2 (synonyms: ω-amidase); PLP,

pyridoxal 5′-phosphate; RT-PCR, reverse transcriptase-

polymerase chain reaction; TCA, tricarboxylic acid;

THA, 2′,A′,6′-Trihydroxyacetophenone; TS, transi-

tion state; UC, ulcerative colitis; UMOD, uromodulin;

UPLC-MS/MS, ultraperformance liquid chromatography-

tandem mass spectrometry; yNit2, yeast nitrilase like

protein2.

Acknowledgments. We thank Dr. Niklas Müller

and Dr. Flávia Rezende (Institute for Cardiovascular

Physiology, Goethe University, Frankfurt am Main,

Germany and German Center of Cardiovascular Re-

search (DZHK), Partner Site Rhein Main, Germany) for

helpful discussions.

COOPER, DENTON1674

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Contributions. AJLC and TTD carried out a con-

siderable amount of work on the GTωA pathway de-

scribed in this review. AJLC wrote the first draft of

the manuscript. TTD drew the figures. Both authors

contributed to and approved the final version of the

manuscript.

Funding. This work was supported by ongoing in-

stitutional funding. No additional grants to carry out

or direct this particular research were obtained.

Ethics declarations. TTD, in association with

Dr.Albert Li, has created a Limited Liability Company

(LiT Biosciences, Spokane, WA, USA) to commercialize

products related to the GTωA pathway. However, LiT

Biosciences did not contribute funding to the writ-

ing of the current review or to any work previously

carried out by the authors. Thus, the authors declare

noconflict of interest.

Open Access. This article is licensed under a Cre-

ative Commons Attribution 4.0 International License,

which permits use, sharing, adaptation, distribution,

and reproduction in any medium or format, as long

as you give appropriate credit to the original author(s)

and the source, provide a link to the Creative Com-

mons license, and indicate if changes were made.

Theimages or other third party material in this article

are included in the article’s Creative Commons license,

unless indicated otherwise in a credit line to the mate-

rial. If material is not included in the article’s Creative

Commons license and your intended use is not permit-

ted by statutory regulation or exceeds the permitted

use, you will need to obtain permission directly from

the copyright holder. To view a copy of this license,

visit http://creativecommons.org/licenses/by/4.0/.

REFERENCES

1. Cooper, A.J., and Meister,A. (1989) An appreciation

of Professor Alexander E. Braunstein. The discovery

and scope of enzymatic transamination, Biochimie, 71,

387-404, https://doi.org/10.1016/0300-9084(89)90169-7.

2. Ovchinnikov, Y. A., Egorov, C. A., Aldanova, N. A.,

Feigina, M. Y., Lipkin, V. M., Abdulaev, N.G., Grishin,

E.V., Kiselev, A.P., Modyanov, N.N., Braunstein, A.E.,

Polyanovsky, O.L., and Nosikov, V.V. (1973) The com-

plete amino acid sequence of cytoplasmic aspartate

aminotransferase from pig heart, FEBS Lett., 29, 31-34,

https://doi.org/10.1016/0014-5793(73)80008-0.

3. Braunstein, A. E. (1957) Les Voies Principales De L’as-

similation Et Dissimilation De L’Azote Chez Les Ani-

maux, in Advances in Enzymology and Related Areas

of Molecular Biology, pp. 335-389, https://doi.org/

10.1002/9780470122648.ch5.

4. Brosnan, J.T. (2000) Glutamate, at the interface between

amino acid and carbohydrate metabolism, J.Nutr., 130,

988S-990S, https://doi.org/10.1093/jn/130.4.988S.

5. Snell, E. E. (1993) From bacterial nutrition to en-

zyme structure: a personal odyssey, Annu. Rev. Bio-

chem., 62, 1-26, https://doi.org/10.1146/annurev.bi.

62.070193.000245.

6. Snell, E. E., and Jenkins, W. T. (1959) The mecha-

nism of the transamination reaction, J. Cell. Com-

par. Physiol., 54, 161-177, https://doi.org/10.1002/jcp.

1030540413.

7. Meister, A. (1955) Transamination, Adv. Enzymol.

Relat. Subj. Biochem., 16, 185-246, https://doi.org/

10.1002/9780470122617.ch4.

8. Meister, A. (1990) On the transamination of en-

zymes, Ann. NY Acad. Sci., 585, 13-31, https://doi.org/

10.1111/j.1749-6632.1990.tb28038.x.

9. Meister,A., and Tice, S.V. (1950) Transamination from

glutamine to alpha-keto acids, J.Biol. Chem., 187, 173-

187, https://doi.org/10.1016/S0021-9258(19)50942-5.

10. Meister, A., Sober, H. A., Tice, S. V., and Fraser, P. E.

(1952) Transamination and associated deamidation of

asparagine and glutamine, J.Biol. Chem., 197, 319-330,

https://doi.org/10.1016/S0021-9258(18)55681-7.

11. Meister,A. (1953) Preparation of enzymatic reactions

of the keto analogues of asparagine and glutamine,

J. Biol. Chem., 200, 571-589, https://doi.org/10.1016/

S0021-9258(18)71403-8.

12. Meister, A., and Otani, T. T. (1957) Omega-Amide and

omega-amino acid derivatives of alpha-ketoglutar-

ic and oxalacetic acids, J. Biol. Chem., 224, 137-148,

https://doi.org/10.1016/S0021-9258(18)65016-1.

13. Cooper, A.J.L., Dorai,T., Pinto, J.T., and Denton, T.T.

(2023) Metabolic heterogeneity, plasticity, and adapta-

tion to “glutamine addiction” in cancer cells: the role

of glutaminase and the GTωA [glutamine transami-

nase-ω-amidase (glutaminase II)] pathway, Biology,

12, 1131, https://doi.org/10.3390/biology12081131.

14. Copper, A. J. L., and Meister, A. (1972) Isolation and

properties of highly purified glutamine transaminase,

Biochemistry, 11, 661-671, https://doi.org/10.1021/

bi00755a001.

15. Cooper, A. J. L., and Meister, A. (1974) Isolation and

properties of a new glutamine transaminase from

rat kidney, J. Biol. Chem., 249, 2554-2561, https://

doi.org/10.1016/S0021-9258(19)42765-8.

16. Hersh, L. B. (1971) Rat liver omega-amidase. Purifi-

cation and properties, Biochemistry, 10, 2884-2891,

https://doi.org/10.1021/bi00791a014.

17. Hersh, L.B. (1972) Rat liver-amidase. Kinetic evidence

for an acyl-enzyme intermediate, Biochemistry, 11,

2251-2256, https://doi.org/10.1021/bi00762a007.

18. Malherbe,P., Alberati-Giani,D., Köhler,C., and Cesura,

A.M. (1995) Identification of a mitochondrial form of

kynurenine aminotransferase/glutamine transami-

naseK from rat brain, FEBS Lett., 367, 141-144, https://

doi.org/10.1016/0014-5793(95)00546-l.

19. Han, Q., Li,J., and Li, J. (2004) pH dependence, sub-

strate specificity and inhibition of human kynurenine

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1675

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

aminotransferase I, Eur. J. Biochem., 271, 4804-4814,

https://doi.org/10.1111/j.1432-1033.2004.04446.x.

20. Han, Q., Robinson,H., Cai, T., Tagle, D.A., and Li, J.

(2009) Biochemical and structural properties of mouse

kynurenine aminotransferase III, Mol. Cell. Biol., 29,

784-793, https://doi.org/10.1128/mcb.01272-08.

21. Pinto, J. T., Krasnikov, B. F., Alcutt, S., Jones, M. E.,

Dorai,T., Villar, M. T., Artigues, A., Li, J., and Cooper,

A.J. (2014) Kynurenine aminotransferase III and glu-

tamine transaminase L are identical enzymes that

have cysteine S-conjugate β-lyase activity and can

transaminate L-selenomethionine, J. Biol. Chem., 289,

30950-30961, https://doi.org/10.1074/jbc.M114.591461.

22. Han, Q., Robinson,H., Cai, T., Tagle, D.A., and Li, J.

(2011) Biochemical and structural characterization

of mouse mitochondrial aspartate aminotransferase,

a newly identified kynurenine aminotransferase-IV,

Biosci. Rep., 31, 323-332, https://doi.org/10.1042/

bsr20100117.

23. URL: https://www.uniprot.org/uniprotkb/Q16773/entry.

24. URL: https://www.uniprot.org/uniprotkb/Q6YP21/entry.

25. Blanco Ayala, T., Lugo Huitrón, R., Carmona Apari-

cio, L., Ramírez Ortega, D., González Esquivel, D.,

Pedraza Chaverrí, J., Pérez de la Cruz, G., Ríos, C.,

Schwarcz, R., and Pérez de la Cruz, V. (2015) Alter-

native kynurenic acid synthesis routes studied in the

rat cerebellum, Front. Cell. Neurosci., 9, 178, https://

doi.org/10.3389/fncel.2015.00178.

26. Yu,P., Di Prospero, N.A., Sapko, M.T., Cai,T., Chen,A.,

Melendez-Ferro,M., Du,F., Whetsell, W.O.,Jr., Guidet-

ti, P., Schwarcz, R., and Tagle, D. A. (2004) Biochem-

ical and phenotypic abnormalities in kynurenine

aminotransferase II-deficient mice, Mol. Cell. Biol.,

24, 6919-6930, https://doi.org/10.1128/mcb.24.16.6919-

6930.2004.

27. Cruzat,V., Macedo Rogero,M., Noel Keane,K., Curi,R.,

and Newsholme, P. (2018) Glutamine: metabolism

and immune function, supplementation and clinical

translation, Nutrients, 10, 1564, https://doi.org/10.3390/

nu10111564.

28. Ohta,Y., Kubo,H., Yashiro,K., Ohashi,K., Tsuzuki,Y.,

Wada,N., Yamamoto,Y., and Saito,K. (2017) Effect of

water-immersion restraint stress on tryptophan catab-

olism through the kynurenine pathway in rat tissues,

J. Physiol. Sci., 67, 361-372, https://doi.org/10.1007/

s12576-016-0467-y.

29. Commandeur, J.N.M., Andreadou,I., Rooseboom,M.,

Out, M., de Leur, L. J., Groot, E., and Vermeulen,

N.P.E. (2000) Bioactivation of selenocysteine se-conju-

gates by a highly purified rat renal cysteine conjugate

β-lyase/glutamine transaminaseK, J.Pharmacol. Exp.

Ther., 294, 753-761.

30. Cooper, A.J., Krasnikov, B.F., Niatsetskaya, Z.V., Pinto,

J.T., Callery, P. S., Villar, M.T., Artigues, A., and Brus-

chi, S.A. (2011) Cysteine S-conjugate β-lyases: import-

ant roles in the metabolism of naturally occurring sul-

fur and selenium-containing compounds, xenobiotics

and anticancer agents, Amino Acids, 41, 7-27, https://

doi.org/10.1007/s00726-010-0552-0.

31. Cooper, A.J., Pinto, J. T., Krasnikov, B. F., Niatsetska-

ya, Z.V., Han,Q., Li,J., Vauzour,D., and Spencer, J. P.

(2008) Substrate specificity of human glutamine trans-

aminase K as an aminotransferase and as a cysteine

S-conjugate beta-lyase, Arch. Biochem. Biophys., 474,

72-81, https://doi.org/10.1016/j.abb.2008.02.038.

32. Lash, L.H., Nelson, R.M., Van Dyke, R.A., and Anders,

M.W. (1990) Purification and characterization of hu-

man kidney cytosolic cysteine conjugate beta-lyase ac-

tivity, Drug Metab. Dispos. Biol. Fate Chem., 18, 50-54.

33. Ellens, K.W., Richardson, L.G.L., Frelin,O., Collins,J.,

Ribeiro, C. L., Hsieh, Y.-f., Mullen, R.T., and Hanson,

A. D. (2015) Evidence that glutamine transaminase

and omega-amidase potentially act in tandem to close

the methionine salvage cycle in bacteria and plants,

Phytochemistry, 113, 160-169, https://doi.org/10.1016/

j.phytochem.2014.04.012.

34. Caligiore, F., Zangelmi, E., Vetro, C., Kentache, T.,

Dewulf, J.P., Veiga-da-Cunha,M., Van Schaftingen, E.,

Bommer,G., and Peracchi,A. (2022) Human cytosolic

transaminases: side activities and patterns of discrim-

ination towards physiologically available alternative

substrates, Cell. Mol. Life Sci., 79, 421, https://doi.org/

10.1007/s00018-022-04439-3.

35. Han, Q., Yang, C., Lu, J., Zhang, Y., and Li, J. (2019)

Metabolism of oxalate in humans: a potential role ky-

nurenine aminotransferase/glutamine transaminase/

cysteine conjugate betalyase plays in hyperoxaluria,

Curr. Med. Chem., 26, 4944-4963, https://doi.org/10.217

4/0929867326666190325095223.

36. Cooper,A., Ginos, J.Z., and Meister, A. (1983) Synthe-

sis and properties of the α-keto acids, Chem. Rev., 83,

321-358, https://doi.org/10.1021/cr00055a004.

37. Herrle, N., Malacarne, P. F., Warwick, T., Cabrera-

Orefice, A., Chen, Y., Gheisari, M., Chatterjee, S.,

Leisegang, M. S., Sarakpi, T., Wionski, S., Lopez, M.,

Koch, I., Keßler, M., Klein, S., Uschner, F. E., Trebic-

ka, J., Brunst, S., Proschak, E., Günther, S., Rosas-

Lemus, M., et al. (2024) The transaminase-ω-amidase

pathway is a redox switch in glutamine metabo-

lism that generates α-ketoglutarate, bioRxiv, https://

doi.org/10.1101/2024.08.28.610061.

38. Cooper, A. J., Shurubor, Y. I., Dorai, T., Pinto, J. T.,

Isakova, E.P., Deryabina, Y.I., Denton, T.T., and Kras-

nikov, B. F. (2016) ω-Amidase: an underappreciated,

but important enzyme in L-glutamine and L-aspar-

agine metabolism; relevance to sulfur and nitrogen

metabolism, tumor biology and hyperammonemic

diseases, Amino Acids, 48, 1-20, https://doi.org/10.1007/

s00726-015-2061-7.

39. Krasnikov, B.F., Nostramo,R., Pinto, J.T., and Cooper,

A.J. (2009) Assay and purification of omega- amidase/

Nit2, a ubiquitously expressed putative tumor

COOPER, DENTON1676

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

suppressor, that catalyzes the deamidation of the

alpha-keto acid analogues of glutamine and aspar-

agine, Anal. Biochem., 391, 144-150, https://doi.org/

10.1016/j.ab.2009.05.025.

40. Cooper, A. J. L. (1988) Glutamine aminotransferases

and ω-amidases, in Glutamine and Glutamate in Mam-

mals, CRC Press, Inc., Boca Raton, Florida, pp.33-52,

https://doi.org/10.1201/9781351072298-4.

41. Dorai,T., Dorai,B., Pinto, J.T., Grasso,M., and Cooper,

A. J. L. (2019) High levels of glutaminase II pathway

enzymes in normal and cancerous prostate suggest

a role in ‘glutamine addiction’, Biomolecules, 10, 2,

https://doi.org/10.3390/biom10010002.

42. Lin, C.-H., Chung, M.-Y., Chen, W.-B., and Chien, C.-H.

(2007) Growth inhibitory effect of the human NIT2

gene and its allelic imbalance in cancers, FEBS J.,

274, 2946-2956, https://doi.org/10.1111/j.1742-4658.

2007.05828.x.

43. GeneCards (2024) NIT2 Gene – Nitrilase Family

Member 2, URL: https://www.genecards.org/cgi-bin/

carddisp.pl?gene=NIT2.

44. Mora,J. (1990) Glutamine metabolism and cycling in

Neurospora crassa, Microbiol. Rev., 54, 293-304, https://

doi.org/10.1128/mr.54.3.293-304.1990.

45. Calderón,J., Morett,E., and Mora,J. (1985) Omega-ami-

dase pathway in the degradation of glutamine in

Neurospora crassa, J.Bacteriol., 161, 807-809, https://

doi.org/10.1128/jb.161.2.807-809.1985.

46. Ta, T. C., Joy, K. W., and Ireland, R. J. (1985) Role of

asparagine in the photorespiratory nitrogen metabo-

lism of pea leaves, Plant Physiol., 78, 334-337, https://

doi.org/10.1104/pp.78.2.334.

47. Zhang, Q., and Marsolais,F. (2014) Identification and

characterization of omega-amidase as an enzyme

metabolically linked to asparagine transamination

in Arabidopsis, Phytochemistry, 99, 36-43, https://

doi.org/10.1016/j.phytochem.2013.12.020.

48. Cobzaru, C., Ganas, P., Mihasan, M., Schleberger, P.,

and Brandsch, R. (2011) Homologous gene clusters

of nicotine catabolism, including a new ω-amidase

for α-ketoglutaramate, in species of three genera of

Gram-positive bacteria, Res. Microbiol., 162, 285-291,

https://doi.org/10.1016/j.resmic.2011.01.001.

49. Jaisson, S., Veiga-da-Cunha, M., and Van Schaftin-

gen, E. (2009) Molecular identification of ω-amidase,

the enzyme that is functionally coupled with gluta-

mine transaminases, as the putative tumor suppressor

Nit2, Biochimie, 91, 1066-1071, https://doi.org/10.1016/

j.biochi.2009.07.002.

50. Krasnikov, B. F., Chien, C. H., Nostramo, R., Pinto,

J.T., Nieves,E., Callaway,M., Sun,J., Huebner,K., and

Cooper, A. J. (2009) Identification of the putative tu-

mor suppressor Nit2 as omega-amidase, an enzyme

metabolically linked to glutamine and asparagine

transamination, Biochimie, 91, 1072-1080, https://

doi.org/10.1016/j.biochi.2009.07.003.

51. Q9NQR4 NIT2_HUMAN, URL: https://www.uniprot.org/

uniprotkb/Q9NQR4/entry#structure.

52. Chien, C.-H., Gao, Q.-Z., Cooper, A.J.L., Lyu, J.-H., and

Sheu, S.-Y. (2012) Structural insights into the catalyt-

ic active site and activity of human Nit2/ω-amidase:

kinetic assay and molecular dynamics simulation,

J.Biol. Chem., 287, 25715-25726, https://doi.org/10.1074/

jbc.M111.259119.

53. Silva Teixeira, C. S., Sousa, S. F., and Cerqueira, N.

(2021) An unsual Cys-Glu-Lys catalytic triad is respon-

sible for the catalytic mechanism of the nitrilase su-

perfamily: a QM/MM study on Nit2, Chemphyschem,

22, 796-804, https://doi.org/10.1002/cphc.202000751.

54. Cooper, A. J. L., and Meister, A. (1981) Comparative

studies of glutamine transaminases from rat tissues,

Compar. Biochem. Physiol. B, 69, 137-145, https://

doi.org/10.1016/0305-0491(81)90223-6.

55. Urinary Exosome Protein Database, URL: https://

esbl.nhlbi.nih.gov/UrinaryExosomes/.

56. PhosphoSitePlus, URL : https://www.phosphosite.org/

proteinAction?id=4207&showAllSites=true.

57. Duthie, S.J., Mavrommatis,Y., Rucklidge,G., Reid,M.,

Duncan, G., Moyer, M. P., Pirie, L. P., and Bestwick,

C. S. (2008) The response of human colonocytes to

folate deficiency in vitro: functional and proteom-

ic analyses, J. Proteome Res., 7, 3254-3266, https://

doi.org/10.1021/pr700751y.

58. Wang,D., Jensen, R.H., Williams, K.E., and Pallavicini,

M. G. (2004) Differential protein expression in MCF7

breast cancer cells transfected with ErbB2, neomycin

resistance and luciferase plus yellow fluorescent pro-

tein, Proteomics, 4, 2175-2183, https://doi.org/10.1002/

pmic.200300728.

59. Pace, H. C., and Brenner, C. (2001) The nitrilase su-

perfamily: classification, structure and function, Ge-

nome Biol., 2, Reviews0001, https://doi.org/10.1186/

gb-2001-2-1-reviews0001.

60. Brenner,C. (2002) Catalysis in the nitrilase superfam-

ily, Curr. Opin. Struct. Biol., 12, 775-782, https://doi.org/

10.1016/s0959-440x(02)00387-1.

61. Peracchi, A., Veiga-da-Cunha, M., Kuhara, T., Ellens,

K.W., Paczia,N., Stroobant,V., Seliga, A.K., Marlaire,S.,

Jaisson,S., Bommer, G.T., Sun,J., Huebner,K., Linster,

C. L., Cooper, A. J. L., and Van Schaftingen, E. (2017)

Nit1 is a metabolite repair enzyme that hydrolyzes de-

aminated glutathione, Proc. Natl. Acad. Sci. USA, 114,

E3233-E3242, https://doi.org/10.1073/pnas.1613736114.

62. Weber, B.W., Kimani, S.W., Varsani,A., Cowan, D.A.,

Hunter, R., Venter, G. A., Gumbart, J. C., and Sewell,

B. T. (2013) The mechanism of the amidases: mutat-

ing the glutamate adjacent to the catalytic triad in-

activates the enzyme due to substrate mispositioning,

J.Biol. Chem., 288, 28514-28523, https://doi.org/10.1074/

jbc.M113.503284.

63. Makumire, S., Su, S., Weber, B. W., Woodward, J. D.,

Wangari Kimani,S., Hunter,R., and Sewell, B.T. (2022)

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1677

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

The structures of the C146A variant of the amidase

from Pyrococcus horikoshii bound to glutaramide

and acetamide suggest the basis of amide recognition,

J. Struct. Biol., 214, 107859, https://doi.org/10.1016/

j.jsb.2022.107859.

64. Liu, H., Gao, Y., Zhang, M., Qiu, X., Cooper, A. J. L.,

Niu, L., and Teng, M. (2013) Structures of enzyme-

intermediate complexes of yeast Nit2: insights into its

catalytic mechanism and different substrate specifici-

ty compared with mammalian Nit2, Acta Crystallogr. D

Biol. Crystallogr., 69, 1470-1481, https://doi.org/10.1107/

s0907444913009347.

65. Pace, H. C., Hodawadekar, S. C., Draganescu, A.,

Huang, J., Bieganowski, P., Pekarsky,Y., Croce, C. M.,

and Brenner, C. (2000) Crystal structure of the worm

NitFhit Rosetta Stone protein reveals a Nit tetram-

er binding two Fhit dimers, Curr. Biol., 10, 907-917,

https://doi.org/10.1016/s0960-9822(00)00621-7.

66. Barglow, K. T., Saikatendu, K. S., Bracey, M. H.,

Huey,R., Morris, G.M., Olson, A.J., Stevens, R.C., and

Cravatt, B.F. (2008) Functional proteomic and struc-

tural insights into molecular recognition in the ni-

trilase family enzymes, Biochemistry, 47, 13514-13523,

https://doi.org/10.1021/bi801786y.

67. Burakoff,R., Chao,S., Perencevich,M., Ying,J., Fried-

man,S., Makrauer,F., Odze,R., Khurana,H., and Liew,

C.-C. (2011) Blood-based biomarkers can differentiate

ulcerative colitis from Crohn’s disease and noninflam-

matory diarrhea, Inflamm. Bowel Dis., 17, 1719-1725,

https://doi.org/10.1002/ibd.21574.

68. Myung, J. K., Gulesserian, T., Fountoulakis, M., and

Lubec, G. (2003) Deranged hypothetical proteins Rik

protein, Nit protein 2 and mitochondrial inner mem-

brane protein, Mitofilin, in fetal Down syndrome

brain, Cell Mol. Biol., 49, 739-746.

69. Semba,S., Han, S.Y., Qin, H.R., McCorkell, K.A., Iliop-

oulos, D., Pekarsky, Y., Druck, T., Trapasso,F., Croce,

C. M., and Huebner, K. (2006) Biological functions

of mammalian Nit1, the counterpart of the inverte-

brate NitFhit Rosetta stone protein, a possible tumor

suppressor, J. Biol. Chem., 281, 28244-28253, https://

doi.org/10.1074/jbc.M603590200.

70. Lin, C., Zhang,J., Lu, Y., Li, X., Zhang, W., Zhang, W.,

Lin, W., Zheng, L., and Li, X. (2018) NIT1 suppresses

tumour proliferation by activating the TGFβ1–Smad2/3

signalling pathway in colorectal cancer, Cell Death &

Disease, 9, 263, https://doi.org/10.1038/s41419-018-0333-3.

71. Mittag,S., Wetzel,F., Müller, S.Y., and Huber,O. (2023)

The Rosetta Stone hypothesis-based interaction of the

tumor suppressor proteins Nit1 and Fhit, Cells, 12,

353, https://doi.org/10.3390/cells12030353.

72. Zheng, B. a., Chai,R., and Yu, X. (2015) Downregula-

tion of NIT2 inhibits colon cancer cell proliferation

and induces cell cycle arrest through the caspase-3

and PARP pathways, Int.J. Mol. Med., 35, 1317-1322,

https://doi.org/10.3892/ijmm.2015.2125.

73. Chen, S., Wang, Z., and Feng,C. (2020) NIT2 overex-

pression predicts poor prognosis in tongue squamous

cell carcinoma patients, Mol. Biol. Rep., 47, 1553-1561,

https://doi.org/10.1007/s11033-019-05197-5.

74. Feng,S., Aplin, C., Nguyen, T.-T. T., Milano, S. K., and

Cerione, R. A. (2024) Filament formation drives ca-

talysis by glutaminase enzymes important in cancer

progression, Nat. Commun., 15, 1971, https://doi.org/

10.1038/s41467-024-46351-3.

75. Buczkowska, J., and Szeliga, M. (2023) Two faces of

glutaminase GLS2 in carcinogenesis, Cancers, 15,

5566, https://doi.org/10.3390/cancers15235566.

76. Ferreira, I. M., Quesñay, J. E. N., Bastos, A. C. S.,

Rodrigues, C. T., Vollmar, M., Krojer, T., Strain-

Damerell,C., Burgess-Brown, N.A., von Delft,F., Yue,

W. W., Dias, S. M. G., and Ambrosio, A. L. B. (2021)

Structure and activation mechanism of the human

liver-type glutaminase GLS2, Biochimie, 185, 96-104,

https://doi.org/10.1016/j.biochi.2021.03.009.

77. Martín-Rufián,M., Tosina,M., Campos-Sandoval, J.A.,

Manzanares, E., Lobo, C., Segura, J. A., Alonso, F. J.,

Matés, J. M., and Márquez, J. (2012) Mammalian glu-

taminase Gls2 gene encodes two functional alterna-

tive transcripts by a surrogate promoter usage mech-

anism, PLoS One, 7, e38380, https://doi.org/10.1371/

journal.pone.0038380.

78. Katt, W. P., Lukey, M. J., and Cerione, R. A. (2017) A

tale of two glutaminases: homologous enzymes with

distinct roles in tumorigenesis, Fut. Med. Chem., 9,

223-243, https://doi.org/10.4155/fmc-2016-0190.

79. Park, C.K., and Horton, N.C. (2019) Structures, func-

tions, and mechanisms of filament forming enzymes:

a renaissance of enzyme filamentation, Biophys.

Rev., 11, 927-994, https://doi.org/10.1007/s12551-019-

00602-6.

80. Park, C.K., and Horton, N.C. (2020) Novel insights into

filament-forming enzymes, Nat. Rev. Mol. Cell Biol.,

21, 1-2, https://doi.org/10.1038/s41580-019-0188-1.

81. Nguyen, T.-T. T., Ramachandran, S., Hill, M. J., and

Cerione, R. A. (2022) High-resolution structures of

mitochondrial glutaminase C tetramers indicate con-

formational changes upon phosphate binding, J.Biol.

Chem., 298, 101564, https://doi.org/10.1016/j.jbc.2022.

101564.

82. Jiang, B., Zhang, J., Zhao, G., Liu, M., Hu, J., Lin, F.,

Wang, J., Zhao, W., Ma,H., Zhang, C., Wu, C., Yao, L.,

Liu,Q., Chen,X., Cao,Y., Zheng,Y., Zhang,C., Han,A.,

Lin,D., and Li,Q. (2022) Filamentous GLS1 promotes

ROS-induced apoptosis upon glutamine depriva-

tion via insufficient asparagine synthesis, Mol. Cell,

82, 1821-1835.e1826, https://doi.org/10.1016/j.molcel.

2022.03.016.

83. Adamoski,D., Dias, M. M., Quesñay, J. E.N., Yang, Z.,

Zagoriy, I., Steyer, A. M., Rodrigues, C. T., da Silva

Bastos, A.C., da Silva, B.N., Costa, R.K.E., de Abreu,

F. M. O., Islam, Z., Cassago, A., van Heel, M. G.,

COOPER, DENTON1678

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

Consonni, S.R., Mattei,S., Mahamid,J., Portugal, R.V.,

Ambrosio, A.L.B., and Dias, S.M.G. (2023) Molecular

mechanism of glutaminase activation through fila-

mentation and the role of filaments in mitophagy pro-

tection, Nat. Struct. Mol. Biol., 30, 1902-1912, https://

doi.org/10.1038/s41594-023-01118-0.

84. Harding, J.J., Telli,M., Munster,P., Voss, M.H., Infan-

te, J. R., DeMichele, A., Dunphy,M., Le, M. H., Molin-

eaux,C., Orford,K., Parlati,F., Whiting, S.H., Bennett,

M. K., Tannir, N. M., and Meric-Bernstam, F. (2021)

A phase I dose-escalation and expansion study of

telaglenastat in patients with advanced or metastatic

solid tumors, Clin. Cancer Res., 27, 4994-5003, https://

doi.org/10.1158/1078-0432.ccr-21-1204.

85. Meric-Bernstam, F., Tannir, N. M., Iliopoulos, O.,

Lee, R. J., Telli, M.L., Fan, A. C., DeMichele,A., Haas,

N. B., Patel, M. R., Harding, J. J., Voss, M. H., Owon-

ikoko, T. K., Carthon,B., Srinivasan, R., Bendell, J. C.,

Jenkins, Y., Whiting, S.H., Orford, K., Bennett, M. K.,

and Bauer, T. M. (2022) Telaglenastat plus cabozan-

tinib or everolimus for advanced or metastatic renal

cell carcinoma: an open-label phaseI trial, Clin. Can-

cer Res., 28, 1540-1548, https://doi.org/10.1158/1078-

0432.ccr-21-2972.

86. Lee, C. H., Motzer, R., Emamekhoo, H., Matrana, M.,

Percent,I., Hsieh, J.J., Hussain,A., Vaishampayan,U.,

Liu, S., McCune, S., Patel, V., Shaheen, M., Bendell, J.,

Fan, A. C., Gartrell, B. A., Goodman, O. B., Nikolina-

kos, P.G., Kalebasty, A.R., Zakharia,Y., Zhang,Z., etal.

(2022) Telaglenastat plus everolimus in advanced re-

nal cell carcinoma: a randomized, double-blinded,

placebo-controlled, phaseII entrata trial, Clin. Cancer

Res., 28, 3248-3255, https://doi.org/10.1158/1078-0432.

ccr-22-0061.

87. Tannir, N. M., Agarwal, N., Porta, C., Lawrence, N. J.,

Motzer,R., McGregor,B., Lee, R.J., Jain, R.K., Davis,N.,

Appleman, L. J., Goodman,O., Jr., Stadler, W. M., Gand-

hi, S., Geynisman, D. M., Iacovelli, R., Mellado, B.,

Sepúlveda Sánchez, J. M., Figlin, R., Powles, T., Akel-

la, L., et al. (2022) Efficacy and safety of telaglenas-

tat plus cabozantinib vs placebo plus cabozantinib

in patients with advanced renal cell carcinoma: the

CANTATA randomized clinical trial, JAMA Oncol., 8,

1411-1418, https://doi.org/10.1001/jamaoncol.2022.3511.

88. Cooper, A. J. L., Dorai, T., Pinto, J. T., and Denton,

T. T. (2022) α-Ketoglutaramate – a key metabolite

contributing to glutamine addiction in cancer cells,

Front. Med., 13, 1035335, https://doi.org/10.3389/

fmed.2022.1035335.

89. Udupa, S., Nguyen, S., Hoang, G., Nguyen, T.,

Quinones, A., Pham, K., Asaka, R., Nguyen, K.,

Zhang, C., Elgogary, A., Jung, J. G., Xu, Q., Fu, J.,

Thomas, A.G., Tsukamoto,T., Hanes,J., Slusher, B.S.,

Cooper, A.J. L., and Le, A. (2019) Upregulation of the

glutaminase II pathway contributes to glutamate

production upon glutaminase I inhibition in pancre-

atic cancer, Proteomics, 19, e1800451, https://doi.org/

10.1002/pmic.201800451.

90. Pham, K., Hanaford, A. R., Poore, B. A., Maxwell,

M. J., Sweeney, H., Parthasarathy, A., Alt, J., Rais, R.,

Slusher, B.S., Eberhart, C.G., and Raabe, E. H. (2022)

Comprehensive metabolic profiling of MYC-amplified

medulloblastoma tumors reveals key dependencies on

amino acid, tricarboxylic acid and hexosamine path-

ways, Cancers (Basel), 14, 1311, https://doi.org/10.3390/

cancers14051311.

91. Ogier, G., Chantepie, J., Deshayes, C., Chantegrel, B.,

Charlot, C., Doutheau, A., and Quash, G. (1993) Con-

tribution of 4-methylthio-2-oxobutanoate and its

transaminase to the growth of methionine-depen-

dent cells in culture. Effect of transaminase inhib-

itors, Biochem. Pharmacol., 45, 1631-1644, https://

doi.org/10.1016/0006-2952(93)90304-f.

92. Quash,G., Roch, A.M., Charlot,C., Chantepie,J., Thom-

as,V., Hamedi-Sangsari,F., and Vila,J. (2004) 4-meth-

ylthio 2-oxobutanoate transaminase: a specific target

for antiproliferative agents, Bull. Cancer, 91, E61-E79.

93. Nematollahi, A., Sun, G., Jayawickrama, G. S., and

Church, W. B. (2016) Kynurenine aminotransferase

isozyme inhibitors: a review, Int. J. Mol. Sci., 17, 946,

https://doi.org/10.3390/ijms17060946.

94. Townsend, D. M., Tew, K. D., He, L., King, J. B., and

Hanigan, M. H. (2009) Role of glutathione S-transfer-

ase Pi in cisplatin-induced nephrotoxicity, Biomed.

Pharmacother., 63, 79-85, https://doi.org/10.1016/

j.biopha.2008.08.004.

95. Cooper, A. J., Bruschi, S. A., Iriarte, A., and Martinez-

Carrion, M. (2002) Mitochondrial aspartate amino-

transferase catalyses cysteine S-conjugate beta-lyase

reactions, Biochem. J., 368, 253-261, https://doi.org/

10.1042/bj20020531.

96. Zhang, L., Cooper, A. J., Krasnikov, B. F., Xu,H., Bub-

ber, P., Pinto, J. T., Gibson, G. E., and Hanigan, M.H.

(2006) Cisplatin-induced toxicity is associated with plat-

inum deposition in mouse kidney mitochondria in vivo

and with selective inactivation of the alpha-ketogluta-

rate dehydrogenase complex in LLC-PK1 cells, Biochem-

istry, 45, 8959-8971, https://doi.org/10.1021/bi060027g.

97. Sukeda,N., Fujigaki, H., Ando,T., Ando, H., Yamamo-

to,Y., and Saito,K. (2023) Identification of 2′,4′,6′-trihy-

droxyacetophenone as promising cysteine conjugate

beta-lyase inhibitor for preventing cisplatin-induced

nephrotoxicity, Mol. Cancer Ther., 22, 873-881, https://

doi.org/10.1158/1535-7163.mct-22-0564.

98. Behar, K. L., Fitzpatrick, S. M., Hetherington, H. P.,

and Shulman, R.G. (1993) Cerebral metabolic studies

in vivo by combined 1H/31P and 1H/13C NMR spec-

troscopic methods, Acta Neurochir. Suppl., 57, 9-20,

https://doi.org/10.1007/978-3-7091-9266-5_2.

99. Vergara,F., Duffy, T.E., and Plum, F. (1973) Alpha-ke-

toglutaramate, a neurotoxic agent in hepatic coma,

Transact. Assoc. Am. Phys., 86, 255-263.

ω-AMIDASE AND α-KETOGLUTARAMATE AS BIOMARKERS 1679

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

100. Vemuganti, R., and Hazell, A. S. (2014) Mechanisms

of hepatic encephalopathy and thiamine deficiency,

Metab. Brain Dis., 29, 889-890, https://doi.org/10.1007/

s11011-014-9579-3.

101. Vergara,F., Plum,F., and Duffy, T.E. (1974) α-ketoglu-

taramate: increased concentrations in the cerebrospi-

nal fluid of patients in hepatic coma, Science, 183, 81-

83, https://doi.org/10.1126/science.183.4120.81.

102. Cooper, A. J. L., Dhar, A.K., Kutt,H., and Duffy, T. E.

(1980) Determination of 2-pyrrolidone-5-carboxyl-

ic and α-ketoglutaramic acids in human cerebro-

spinal fluid by gas chromatography, Anal. Biochem.,

103, 118-126, https://doi.org/10.1016/0003-2697(80)

90245-6.

103. Kuhara, T., Inoue, Y., Ohse, M., Krasnikov, B. F., and

Cooper, A. J. (2011) Urinary 2-hydroxy-5-oxoproline,

the lactam form of α-ketoglutaramate, is marked-

ly increased in urea cycle disorders, Anal. Bioanal.

Chem., 400, 1843-1851, https://doi.org/10.1007/s00216-

011-4688-x.

104. Kuhara,T., Ohse,M., Inoue,Y., and Cooper, A.J. (2011)

A GC/MS-based metabolomic approach for diagnosing

citrin deficiency, Anal. Bioanal. Chem., 400, 1881-1894,

https://doi.org/10.1007/s00216-011-4766-0.

105. Duffy, T.E., Cooper, A.J.L., and Meister,A. (1974) Iden-

tification of α-ketoglutaramate in rat liver, kidney, and

brain: relationship to glutamine transaminase and

ω-amidase activities, J. Biol. Chem., 249, 7603-7606,

https://doi.org/10.1016/S0021-9258(19)81280-2.

106. Shurubor, Y.I., Cooper, A.J., Isakova, E.P., Deryabina,

Y.I., Beal, M. F., and Krasnikov, B.F. (2016) HPLC de-

termination of α-ketoglutaramate [5-amino-2,5-dioxo-

pentanoate] in biological samples, Anal. Biochem., 494,

52-54, https://doi.org/10.1016/j.ab.2015.11.003.

107. Halámková, L., Mailloux, S., Halámek, J., Cooper,

A. J., and Katz, E. (2012) Enzymatic analysis of α-ke-

toglutaramate – a biomarker for hyperammonemia,

Talanta, 100, 7-11, https://doi.org/10.1016/j.talanta.

2012.08.022.

108. Cooper, A. J. L. (1978) Spot test for the detection of

α-ketoglutaramic acid in human cerebrospinal fluid,

Anal. Biochem., 90, 444-446, https://doi.org/10.1016/

0003-2697(78)90049-0.

109. Bray, H.G., James, S.P., Raffan, I.M., and Thorpe, W.V.

(1949) The enzymic hydrolysis of glutamine and its

spontaneous decomposition in buffer solutions, Bio-

chem.J., 44, 625-627.

110. Van der Werf,P., Orlowski,M., and Meister,A. (1971)

Enzymatic conversion of 5-oxo-L-proline (L-pyrroli-

done carboxylate) to L-glutamate coupled with cleav-

age of adenosine triphosphate to adenosine diphos-

phate, a reaction in the -glutamyl cycle, Proc. Natl.

Acad. Sci. USA, 68, 2982-2985, https://doi.org/10.1073/

pnas.68.12.2982.

111. Vannucci, R.C., Brucklacher, R.M., and Vannucci, S.J.

(1999) CSF glutamate during hypoxia-ischemia in the

immature rat, Brain Res. Dev. Brain Res., 118, 147-151,

https://doi.org/10.1016/s0165-3806(99)00142-x.

112. Hagberg,H., Thornberg, E., Blennow, M., Kjellmer, I.,

Lagercrantz, H., Thiringer, K., Hamberger, A., and

Sandberg,M. (1993) Excitatory amino acids in the ce-

rebrospinal fluid of asphyxiated infants: relationship

to hypoxic-ischemic encephalopathy, Acta Paediatrica,

82, 925-929, https://doi.org/10.1111/j.1651-2227.1993.

tb12601.x.

113. Häberle,J. (2013) Clinical and biochemical aspects of

primary and secondary hyperammonemic disorders,

Arch. Biochem. Biophys., 536, 101-108, https://doi.org/

10.1016/j.abb.2013.04.009.

114. Cooper, A. J., and Kuhara, T. (2014) α-Ketoglutara-

mate: an overlooked metabolite of glutamine and a

biomarker for hepatic encephalopathy and inborn er-

rors of the urea cycle, Metab. Brain Dis., 29, 991-1006,

https://doi.org/10.1007/s11011-013-9444-9.

115. Komatsu, M., Tanaka, N., Kimura,T., and Yazaki, M.

(2023) Citrin deficiency: clinical and nutritional fea-

tures, Nutrients, 15, 2284, https://doi.org/10.3390/

nu15102284.

116. Frainay, C., Aros, S., Chazalviel, M., Garcia, T., Vin-

son, F., Weiss, N., Colsch, B., Sedel, F., Thabut, D.,

Junot, C., and Jourdan, F. (2019) MetaboRank: net-

work-based recommendation system to interpret

and enrich metabolomics results, Bioinformatics,

35, 274-283, https://doi.org/10.1093/bioinformatics/

bty577.

117. Bächle, H., Sekula, P., Schlosser, P., Steinbrenner, I.,

Cheng, Y., Kotsis, F., Meiselbach, H., Stockmann, H.,

Schönherr, S., Eckardt, K. U., Devuyst, O., Scher-

berich,J., Köttgen,A., and Schultheiss, U.T. (2023) Uro-

modulin and its association with urinary metabolites:

the German Chronic Kidney Disease Study, Nephrol.

Dial. Transplant., 38, 70-79, https://doi.org/10.1093/

ndt/gfac187.

118. Pruijm, M., Ponte, B., Ackermann, D., Paccaud, F.,

Guessous, I., Ehret, G., Pechère-Bertschi, A., Vogt,B.,

Mohaupt, M. G., Martin, P.Y., Youhanna, S. C., Näge-

le, N., Vollenweider, P., Waeber, G., Burnier, M.,

Devuyst, O., and Bochud, M. (2016) Associations of

urinary uromodulin with clinical characteristics

and markers of tubular function in the general pop-

ulation, Clin. J. Am. Soc. Nephrol., 11, 70-80, https://

doi.org/10.2215/cjn.04230415.

119. Devuyst,O., Olinger, E., and Rampoldi,L. (2017) Uro-

modulin: from physiology to rare and complex kid-

ney disorders, Nat. Rev. Nephrol., 13, 525-544, https://

doi.org/10.1038/nrneph.2017.101.

120. Scherberich, J. E., Gruber, R., Nockher, W. A., Chris-

tensen, E.I., Schmitt,H., Herbst,V., Block,M., Kaden,J.,

and Schlumberger, W. (2018) Serum uromodulin-

a marker of kidney function and renal parenchy-

mal integrity, Nephrol. Dial. Transplant., 33, 284-295,

https://doi.org/10.1093/ndt/gfw422.

COOPER, DENTON1680

BIOCHEMISTRY (Moscow) Vol. 89 No. 10 2024

121. Schaeffer, C., Devuyst, O., and Rampoldi, L. (2021)

Uromodulin: roles in health and disease, Annu. Rev.

Physiol., 83, 477-501, https://doi.org/10.1146/annurev-

physiol-031620-092817.

122. Sidorov, E. V., Bejar, C., Xu, C., Ray, B., Gordon, D.,

Chainakul,J., and Sanghera, D.K. (2021) Novel metab-

olites as potential indicators of ischemic infarction

volume: a pilot study, Translat. Stroke Res., 12, 778-

784, https://doi.org/10.1007/s12975-020-00876-z.

123. Shurubor, Y. I., Rogozhin, A. E., Isakova, E. P., Dery-

abina, Y. I., and Krasnikov, B. F. (2023) Tricarboxylic

acid metabolite imbalance in rats with acute thioac-

etamide-induced hepatic encephalopathy indicates

incomplete recovery, Int.J. Mol. Sci., 24, 1384, https://

doi.org/10.3390/ijms24021384.

124. Barko, P. C., Rubin, S. I., Swanson, K. S., McMichael,

M. A., Ridgway, M. D., and Williams, D.A. (2023) Un-

targeted analysis of serum metabolomes in dogs with

exocrine pancreatic insufficiency, Animals, 14, 2313,

https://doi.org/10.3390/ani13142313.

125. Pletcher, R.C., Hardman, S.L., Intagliata, S.F., Lawson,

R. L., Page, A., and Tennessen, J. M. (2019) A genetic

screen using the drosophila melanogaster trip rnai

collection to identify metabolic enzymes required

for eye development, G3, 9, 2061-2070, https://doi.org/

10.1534/g3.119.400193.

126. Rotelli, M. D., Bolling, A. M., Killion, A. W., Weinberg,

A.J., Dixon, M.J., and Calvi, B.R. (2019) An RNAi screen

for genes required for growth of drosophila wing tissue,

G3, 9, 3087-3100, https://doi.org/10.1534/g3.119.400581.

127. Unkefer, P.J., Knight, T. J., and Martinez, R. A. (2023)

The intermediate in a nitrate-responsive ω-amidase

pathway in plants may signal ammonium assimila-

tion status, Plant Physiol., 191, 715-728, https://doi.org/

10.1093/plphys/kiac501.

128. Shen,D., Kruger, L., Deatherage,T., and Denton, T. T.

(2020) Synthesis of α-ketoglutaramic acid, Anal. Bio-

chem., 607, 113862, https://doi.org/10.1016/j.ab.2020.

113862.

129. Martinez, R. A., and Unkefer, P. J. (2001) Preparation of

2-Hydroxy-5-Oxoproline and Analogs Thereof, Patent,

Los Alamos National Security LLC, United States.

130. Denton, T. T., and Cooper, A. J. L. (2023) Chemistry,

biochemistry and clinical relevance of the glutamine

metabolite α-ketoglutaramate/2-hydroxy-5-oxoproline,

Austr. J. Chem., 76, 361-371, https://doi.org/10.1071/

CH22264.

Publisher’s Note. Pleiades Publishing remains

neutral with regard to jurisdictional claims in published

maps and institutional affiliations. AI tools may have

been used in the translation or editing of this article.