Proton nmr studies of substrate hydrogen exchange reactions catalyzed by L-methionine .gamma.-lyase

Abstract

H exchange reactions of various L-amino acids catalyzed by L-methionine .gamma.-lyase of Pseudomonas putida were studied. The enzyme catalyzes the rapid exchange of the .alpha.- and .beta.-H of L-methionine and S-methyl-L-cysteine with deuterium from the solvent. The rate of .alpha.-H exchange was .apprx. 40 times faster than that of the enzymatic elimination reaction of the S-containig amino acids. The enzyme also catalyzes the exchange reaction of .alpha.- and .beta.-H of the following straight-chain L-amino acids which are not susceptible to elimination: norleucine, norvaline, .alpha.-aminobutyrate and alanine. The exchange rates of the .alpha.-H and the total .beta.-H of L-alanine and L-.alpha.-aminobutyrate with deuterium followed 1st-order kinetics. For L-norvaline, L-norleucine, S-methyl-L-cysteine and L-methionine, the rate of .alpha.-H exchange followed 1st-order kinetics, but the rate of total .beta.-H exchange decreased due to a primary isotope effect at the .alpha.-position. One .beta.-H of S-methyl-L-cysteine was exchanged faster than the other, although both the .beta.-H were exchanged completely with deuterium ultimately. L-Phenylalanine and L-tryptophan slowly underwent .alpha.-H exchange. The pro-R H of glycine was deuterated stereospecifically. None of the following amino acids were susceptible to the enzymatic H exchange: D isomers of the above amino acids, branched chain L-amino acids, acidic L-amino acids, and basic L-amino acids. L-Methionine derivatives such as methionine sulfoxide having a good leaving group at the .gamma.-C, which are substrates in .alpha.,.gamma.-elimination, were also not susceptible to the H exchange. In this case, the .beta.2H,.gamma.-2H species of .alpha.-ketobutyrate was exclusively formed. The enzyme catalyzes deamination of L-vinylglycine in 2H2O to give also the same .alpha.-ketobutyrate species. These results are consistent with the proposed mechanism that the .alpha.,.gamma.-elimination proceeds through a Schiff base of vinylglycine with pyridoxal 5''-phosphate.