Reaction mechanism of Escherichia coli cystathionine .gamma.-synthase: direct evidence for a pyridoxamine derivative of vinylgloxylate as a key intermediate in pyridoxal phosphate dependent .gamma.-elimination and .gamma.-replacement reactions

Abstract

Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia. Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.

Evolutionary relationships among pyridoxal‐5′‐phosphate‐dependent enzymes

“…Certain synthases, e.g. tryptophan synthase (Ahmed et al, 1986) and 0-succinylhomoserine (thio1)-lyase (BrzoviC et al, 1990), have been reported to catalyze in the absence of the replacing substrate the corresponding elimination reaction instead of the replacement reaction. The above side reactions correspond to the regio-specificity for the principal reaction except in the case of tryptophan synthase.…”

Kinetics and Inhibition of Recombinant Human Cystathionine γ-Lyase

“…The high resolution crystal structures of Escherichia coli CBL (4) and CGS (5), together with crystallographic (6) and kinetic investigations (6 -9) on inhibitors, allowed the suggestion and evaluation of reaction mechanisms (4,10). Both CBL and CGS are homotetramers composed of ϳ40 -45-kDa subunits and carry one PLP cofactor per monomer covalently bound via a Schiff base to an active site lysine.…”

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

“…At the resolution of our structure, we do not exclude a saturation of the N-CA bond, but the geometry of the complex is more compatible with the double N-CA bond. For mechanisms of ␥-elimination and ␥-replacement reactions catalyzed by PLP-dependent enzymes, it was postulated that ketimine (Scheme 1, V) is a plausible intermediate for effective labilization of the C␤-proton to proceed (17). Its formation in the course of these reactions catalyzed by E. coli cystathionine ␥-synthase was detected by stopped-flow data in the same paper.…”

“…The enzyme is also able to catalyze the ␤-elimination reaction of L-cysteine and the S-substituted L-cysteines (Reaction 2) and the ␥-and ␤-replacement reactions of L-methionine and L-cysteine and their analogs (15,16). The chemical mechanism (Scheme 1) of ␥-elimination reaction was proposed by Brzovic et al (17).…”