Cystathionine 7-synthetase (EC 4.2.99.9), a key enzyme in bacterial methionine biosynthesis, has been found to use l-vinylglycine (2-amino-3-butenoate) and L-β-halo-aminobutyrates (×=F, CI) as substrates in addition to the physiological γ-substituted substrate O-succinyl-l-homoserine (OSHS). Vinylglycine is a substrate both for a-ketobutyrate formation (the normal product from γ elimination with OSHS) and for cystathionine formation (the normal γ-re-placement product with OSHS) in the presence of cysteine. This behavior substantiates that the stabilized vinylglycine-pyridoxal phosphate (PLP) a carbanion is the key partitioning species in this enzyme's catalysis. The Vmax values for ke-tobutyrate production and cystathionine 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 β-haloaminobutyrates undergo catalyzed HX elimination to yield bound aminocrotonate-PLP directly as an initial intermediate and as a precursor of ke-tobutyrate. 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 a carbanion to aminocrotonate appears functionally unidirectional. The HX-elimination step from β-chloroaminobutyrate is non-concerted as demonstrated by a 3H2O ⇌ chloroaminobutyrate exchange reaction. Further suggestion for discrete β-halo-a-carbanionic intermediates derives from the observation that the haloaminobutyrates appear to partition between keto-butyrate 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 β-haloaminobutyrate pathway. The β-halo-a-carbanion-PLP intermediate has β-halo-a-imino-dihydropyridine 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. © 1979, American Chemical Society. All rights reserved.
