Mechanistic studies with vinylglycine and beta-haloaminobutyrates as substrates for cystathionine gamma-synthetase from Salmonella typhimurium

Abstract

Cystathionine gamma-synthetase (EC 4.2.99.9), a key enzyme in bacterial methionoine biosynthesis, has been found to use L-vinylglycine (2-amino-3-butenoate) and L-beta-haloaminobutyrates (X = F, Cl) as substrates in addition to the physiological gamma-substituted substrate O-succinyl-L-homoserine (OSHS). Vinylglycine is a substrate both for alpha-ketobutyrate formation (the normal product from gamma elimination with OSHS) and for cystathionine formation (the normal gamma-replacement product with OSHS) in the presence of cysteine. This behavior substantiates that the stabilized vinylglycine--pyridoxal phosphate (PLP) alpha carbanion is the key partitioning species in this enzyme's catalysis. The Vmax values for ketobutyrate production and cystathonine formation from vinylglycine are equivalent at approximately 45 U/mg, whereas the corresponding Vmax values from OSHS are 20 and 200 U/mg, respectively, suggesting different rate-determining steps with these two substrates. The beta-haloaminobutyrates undergo catalyzed HX elimination to yield bound aminocrotonate--PLP directly as a an initial intermediate and as a precursor of ketobutyrate. Little or no cystathionine formation is detectable when these substrates are incubated with enzyme and the normal cosubstrate cysteine, strongly indicating that the aminocrotonate--PLP intermediate is not in rapid, reversible equilibrium with the stabilized vinylglycine--PLP carbanion; in normal catalysis, the prototropic shift from alpha carbanion to aminocrotonate appears functionally unidirectional. The HX-elimination step from beta-chloroaminobutyrate is nonconcerted as demonstrated by a 3H2O in equilibrium chloroaminobutyrate exchange reaction. Further suggestion for discrete beta-halo-alpha-carbanionic intermediates derives from the observation that the haloaminobutyrates appear to a partition between ketobutyrate formation and enzyme inactivation. Since neither vinylglycine nor OSHS causes any detectable inactivation during turnover, it is likely that the inactivation species is not a common intermediate, i.e., the electrophilic aminocrotonate--PLP species (a potential Michael acceptor), but rather a species peculiar to the beta-haloaminobutyrate pathway. The beta-halo-alpha-carbanion--PLP intermediate has beta-halo-alpha-iminodihydropyridine character in the p-quinoid resonance contributor and is a good candidate for an alkylating agent by an SN2--displacement mechanism. Spectroscopic analyses of incubations with the various amino acid substrates show a number of long-wavelength absorbing species forming during turnover, tentative assignments are suggested.