Mechanistic studies on reactions of bacterial methionine gamma-lyase with olefinic amino acids.

Abstract

Methionine gamma-lyase (EC 4.4.1.11), which catalyzes the formation of methanethiol, alpha-ketobutyrate, and ammonia from L-methionine (eq 1), promotes the oxidative deamination of several four- and five-carbon olefinic amino acids (1-5). With the exception of vinylglycine (1), the Vmax rates of keto acid formation from the unsaturated substrate analogues are substantially lower than that for processing of methionine to alpha-ketobutyrate; vinylglycine is deaminated to ketobutyrate and ammonia with a Vmax twice that for L-methionine turnover. L-Allylglycine, L-2-amino-3-trans-pentenoate, and L-2-amino-3-cis-pentenoate (2, 4, 5) are all converted to 2-keto-pentanoic acid (alpha-ketovalerate). L-2-Amino-3-cis-pentenoate (5) is also a time-dependent, irreversible inactivator of the enzyme. None of the other substrate analogues tested appears to inactivate the enzyme. Spectral analysis of the enzymatic reaction with cis isomer 5 reveals the formation of a high-wavelength chromophore (lambda max = 550 nm ) which implies that a beta, gamma-unsaturated pyridoxal p-quinoid (VI) accumulates. No such absorbing species appears to form during the reaction of trans isomer 4 with methionine gamma-lyase. But a 550-nm chromophore develops when both 4 and 5 are reacted with Al(NO3)3 and pyridoxal methochloride in methanolic KOH. It would appear that the geometry of the protein and the olefinic amino acid as an intermediate enzyme-substrate adduct controls the kinetics of reaction, such that azaallylic isomerization becomes selectively rate determining for reaction with 5. When this isomerization is slow, an accumulating Michael-type acceptor (VI) could lead to the observed irreversible inactivation of the enzyme.