Cystathionine γ-Synthase

One of the reactions catalyzed by cystathionine γ-synthase, a bacterial pyridoxal phosphate enzyme, is the decomposition of O-succinylhomoserine, by γ elimination, to yield ammonia, succinate, and α-ketobutyrate. It had been shown that when this reaction was carried out in 2H2O 1 atom of deuterium was stereospecifically introduced (the absolute configuration is confirmed in this paper) at carbon 3 of α-ketobutyrate, but considerably less than 1 atom of deuterium was present in the newly formed terminal methyl group. The latter result indicated that a portion of the hydrogen introduced at carbon 4 originated in the substrate, presumably by intramolecular proton transfer from either the α or the β position of the homoserine moiety. To answer this question we have synthesized homoserine with deuterium in the α, the β, or in both positions, have succinylated the various preparations, and allowed them to be decomposed in H2O or 2H2O by cystathionine synthase. The distribution of hydrogen (1H) and deuterium in the resultant α-ketobutyrate has been determined by NMR and mass spectroscopy. The results showed that: (a) there was proton (1H) transfer from both α and β positions of substrate to γ position of product; (b) the β hydrogen transferred was the same as the one shown in the preceding paper to be rapidly exchanged; (c) there was not a measurable amount of deuterium transfer from either α or β position. The effects of α and β deuterium substitution on the kinetic constants for the γ elimination reaction were found to be rather small. The explanation postulated for these results involves a single polyhydric basic group of the enzyme functioning in the abstraction of both the α and β hydrogens of the substrate and in a series of intramolecular proton transfers leading to their partial retention in the methyl group of α-ketobutyrate.