Abstract

Kinetic and Spectral Parameters of Interaction of Citrobacter freundii Methionine γ-Lyase with Amino Acids

E. A. Morozova¹, N. P. Bazhulina¹, N. V. Anufrieva¹, D. V. Mamaeva^1,2, Y. V. Tkachev¹, S. A. Streltsov¹, V. P. Timofeev¹, N. G. Faleev^3, and T. V. Demidkina^1

¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, 119991 Moscow, Russia; fax: (499) 135-1405; E-mail: tvd@eimb.ru

²Present address: Institute of Human Genetics, CNRS UPR1142, 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France; fax: +33(0)499-619901; E-mail: daria.mamaeva@mail.ru

³Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, 199991 Moscow, Russia; fax: (499) 135-0551; E-mail: ngfal@ineos.ac.ru

^* To whom correspondence should be addressed.

Received May 28, 2010; Revision received June 20, 2010
Kinetic parameters of Citrobacter freundii methionine γ-lyase were determined with substrates in γ-elimination reactions as well as the inhibition of the enzyme in the γ-elimination of L-methionine by amino acids with different structure. The data indicate an important contribution of the sulfur atom and methylene groups to the efficiency of binding of substrates and inhibitors. The rate constants of the enzyme-catalyzed exchange of C-α- and C-β-protons with deuterium were determined, as well as the kinetic isotope effect of the deuterium label in the C-α-position of inhibitors on the rate of exchange of their β-protons. Neither stereoselectivity in the β-proton exchange nor noticeable α-isotope effect on the exchange rates of β-protons was found. The ionic and tautomeric composition of the external Schiff base of methionine γ-lyase was determined. Spectral characteristics (absorption and circular dichroism spectra) of complexes with substrates and inhibitors were determined. The spectral and kinetic data indicate that deamination of aminocrotonate should be the rate-determining stage of the enzymatic reaction.
KEY WORDS: Citrobacter freundii methionine γ-lyase, pyridoxal 5′-phosphate, substrates, inhibitors, proton exchange, spectral characteristics
DOI: 10.1134/S0006297910100093

Kinetic and Spectral Parameters of Interaction of Citrobacter freundii Methionine γ-Lyase with Amino Acids

E. A. Morozova1, N. P. Bazhulina1, N. V. Anufrieva1, D. V. Mamaeva1,2, Y. V. Tkachev1, S. A. Streltsov1, V. P. Timofeev1, N. G. Faleev3*, and T. V. Demidkina1*

E. A. Morozova¹, N. P. Bazhulina¹, N. V. Anufrieva¹, D. V. Mamaeva^1,2, Y. V. Tkachev¹, S. A. Streltsov¹, V. P. Timofeev¹, N. G. Faleev^3, and T. V. Demidkina^1