U.S. flag

An official website of the United States government

Dot gov

Official websites use .gov
A .gov website belongs to an official government organization in the United States.


Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.


Main content area

The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase

Natalya V. Anufrieva, Nicolai G. Faleev, Elena A. Morozova, Natalia P. Bazhulina, Svetlana V. Revtovich, Vladimir P. Timofeev, Yaroslav V. Tkachev, Alexei D. Nikulin, Tatyana V. Demidkina
Biochimica et biophysica acta 2015 v.1854 no.9 pp. 1220-1228
Citrobacter freundii, X-radiation, absorption, active sites, deuterium oxide, hydrogen bonding, mutants, oxygen, phenylalanine, phosphates, proteins, protons, pyridoxal, pyridoxal phosphate, site-directed mutagenesis, spectral analysis, tyrosine
In the spatial structure of methionine γ-lyase (MGL, EC from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate “handle” of pyridoxal 5′-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.