Mechanism of the Addition Half of the O-Acetylserine Sulfhydrylase-A Reaction

Abstract

O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the beta-acetoxy group of O-acetyl-l-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the alpha-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the alpha-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pK(a) > 7, while the rate constant is pH-independent for substrates with a pK(a) < 7 suggesting that the pK(a)s of the substrate and an enzyme group are important in this half of the reaction. In D(2)O, at low pD values, the amino acid external Schiff base is trapped, while in H(2)O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new beta-substituted amino acids were produced and characterized by (1)H NMR spectroscopy.

Cysteine Synthase (CysM) of Mycobacterium tuberculosis Is an O-Phosphoserine Sulfhydrylase

“…In agreement with the findings of Begley and colleagues (15), we also show that the most efficient and probably natural sulfur donor is thiocarboxylated CysO. The reaction with sulfide is slow compared with bona fide OAS enzymes where the rate of the second half-reaction was determined as Ͼ1000 s Ϫ1 (23). This observation indicates that the aminoacrylate intermediate is not as exposed to spontaneous nucleophilic attack as in OASS-A (CysK) enzymes.…”

“…The kinetic analysis of the individual half-reactions was carried out using the formalism described by Cook and co-workers (14,23). First order rate constants (k obs ) were derived from an exponential fit to the recorded spectrophotometric data.…”

Molecular Basis of Cysteine Biosynthesis in Plants

“…Although titrations of the N77D, Q147A, and Q147E mutants with O-acetylserine yielded no detectable ␣-aminoacrylate intermediate, these mutants still catalyze cysteine formation with 1000-fold lower turnover rates compared with wild-type enzyme. In the OASS reaction, formation of the ␣-aminoacrylate intermediate is the rate-limiting step, and the intermediate decays with time in the absence of sulfur (20,46,47). The lack of detectable intermediate in the N77D, Q147A, and Q147E mutants indicates that the rate of intermediate formation is now slower than the dissociation rate, consistent with an increased K d value for O-acetylserine.…”

“…With regard to sulfide binding, substitution of either amino acid yields mutant enzymes (T74A, T74S, S75A, and S75N) that display more than 10-fold increases in the K m value for sulfide (TABLE TWO). A defined sulfide binding in OASS has not been identified (47); however, the effects of mutating Thr 74 and Ser 75 suggest that these residues play a role in stabilizing the transition state of the second half-reaction. The T74A, S75A, and S75N mutations also affect ligand binding, since titrations of these mutant enzymes showed no detectable signal for the ␣-aminoacrylate intermediate or the external aldimine with cysteine or methionine.…”

The C‐terminal of CysM from Mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity

“…Concentration The kinetic analysis of the individual half-reactions and determination of the kinetic parameters was carried out using the formalism described in [23,27]. First order rate constants (k obs ) were derived from an exponential fit to the recorded time courses.…”