Kinetic and spectral parameters of interaction of Citrobacter freundii methionine γ-lyase with amino acids

Abstract

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.

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

“…In both cases of interaction of MGL, the latest possible intermediate is an enamine (see supplemental Scheme S1). For these amino acids, the existence of exchange of both C␣-and C␤-protons was shown (19). Two discernable steps can be noticed on the absorbance traces.…”

“…Interestingly, the rate constant of the isotopic exchange of C␣-protons for L-alanine is 10 times smaller than that of glycine and L-norvaline (19). The process characterized with the rate constant k 1 should represent the complex mechanism, including not only the multistage process of the Schiff base formation between incoming amino acid and Lys-210, but possibly the enzyme reorganization during the incorporation of the amino acids into the active site.…”

Gene cloning, characterization, and cytotoxic activity of methionine γ‐lyase from Clostridium novyi

“…The band at 500 nm is typical of quinonoid intermediates that occur in reactions catalyzed by PLP‐dependent enzymes . The absorption spectrum of MGLs from C. sporogenes and C. tetani contains a band with a maximum at 505 nm , while this band is barely noticeable in the spectrum of MGL from C. freundii . The spectrum was resolved into bands corresponding to individual electron transitions.…”

“…The parameters of two tautomers and conformers of C. freundii MGL internal aldimine (Fig. ) and three vibronic bands of a quinonoid (Fig. ) were used.…”

“…. The concentration of homogeneous protein was determined by the absorbance at 278 nm, the extinction coefficient ( ) being 0.8 . Protein purity was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under denaturating conditions, and found to be 95%.…”