Inactivation of alpha-Ketoglutarate Dehydrogenase during Its Enzymatic Reaction

V. I. Bunik^1,2 and O. G. Pavlova¹

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 939-0338; E-mail: vbun@bac.genebee.msu.su

²To whom correspondence should be addressed.

Submitted December 25, 1996; revision submitted June 5, 1997.

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The activity of some muscle alpha-ketoglutarate dehydrogenase complexes (KGDC) decreases during their enzymatic reaction as a result of inactivation of the first component of the complex, namely, alpha-ketoglutarate dehydrogenase (KGD). This decrease is associated with transformation of the complex produced by KGD and alpha-keto substrate in the course of oxidative phosphorylation. Kinetics of KGD inactivation during the reaction depends on the keto substrate structure. When alpha-ketoglutarate (KG) is used as a substrate, KGD inactivation occurs in two stages. The major (irreversible) decrease in its activity is observed during the first minutes of reaction. During reaction with a catalytically active KG analog, alpha-ketoadipate (KA), the enzyme is irreversibly inactivated in one stage. This suggests that the reversible stage of inactivation is due to the high rate of catalysis, which is characteristic of KG-utilizing reactions. Decrease in the rate of KG oxidation after treatment of the enzyme with sulfhydryl reagents eliminates this reversible stage. Preincubation of KGD with KG phospho-derivatives (succinylphosphonate or its monomethyl ether) changes the properties of KGD in a similar way to the reversible decrease of activity during catalysis. The arginine residue of KGD, which is essential for enzymatic activity, becomes inaccessible for butanedione in the complexes of KGD with some phospho-derivatives. The data suggest that the reversible inactivation of KGD in the course of catalysis is due to an interaction of the leaving substrate carboxyl with the essential arginine residue of the enzyme.

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KEY WORDS: alpha-ketoglutarate dehydrogenase, alpha-ketoglutarate, alpha-ketoadipate, inactivation during catalysis.

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