Abstract
O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step in the de novo synthesis of L-cysteine in Salmonella typhimurium. Complementary cofactor mutagenesis in which the active site PLP is substituted with cofactor analogs is used to test the mechanism proposed for the OASS. Data obtained with the pyridoxal 5'-deoxymethylenephosphonate-substituted enzyme suggest that the binding of OAS as it forms the external Schiff base is such that the acetate side chain is properly positioned for elimination (orthogonal to the developing alpha,beta-double bond) only about 1% of the time. Data support the assignment of an enzyme group with a pK of 6.7 that interacts with the acetyl side chain, maintaining it orthogonal to the developing alpha,beta-double bond. Similar studies of the 2'-methylpyridoxal 5'-phosphate-substituted enzyme suggest that, although the mechanism is identical to that catalyzed by native OASS, the reaction coordinate for alpha-proton abstraction may be decreased compared with that observed for the native enzyme.
Highlights
The biosynthesis of L-cysteine in Salmonella typhimurium proceeds via a substitution of the -hydroxyl of L-serine with a thiol
A chemical mechanism has been proposed based on pH studies in which the ␣-amine of OAS must be unprotonated for optimum binding and formation of the external Schiff base; an enzyme group hydrogen-bonds the acetyl carbonyl to assist in the elimination of acetate, and the active site lysine that originally participated in the internal Schiff base linkage acts as a general base to abstract the ␣-proton (6)
If the transition state is earlier for the PDMP enzyme based on the assumed synchronous concerted reaction, the above data suggest that the ␣,-elimination reaction of PDMP-O-Acetylserine sulfhydrylase (OASS) is perhaps even more facile than that catalyzed by native OASS. (Even if the reaction is not completely synchronous it is unlikely to affect the interpretation significantly.) The remaining question is why only 1% of the OAS external Schiff base is converted to product at any given time? The answer likely lies in the only difference between the pH profiles for the PDMPOASS and the native enzyme, i.e. the lack of a pH dependence of the V/K for OAS at low pH
Summary
The biosynthesis of L-cysteine in Salmonella typhimurium proceeds via a substitution of the -hydroxyl of L-serine with a thiol. The O-acetyl-L-serine is formed and converted in the presence of sulfide to L-cysteine by O-acetylserine sulfhydrylase (OASS1 EC 4.2.99.8). OASS has a visible max of 412 nm, while the presence of O-acetyl-L-serine results in the formation of an ␣-aminoacrylate intermediate absorbing at 330 and 470 nm (3). Replacement of the native PLP cofactor with analogs has been used in other systems to map the cofactor site and obtain additional information on the reaction mechanism (11–18). The resolution of the PLP cofactor from OASS was achieved by dissociation of the ␣-aminoacrylate intermediate from the active site in the presence of 5 M guanidinium chloride (18). The first two cofactors produce active OASS species, whereas pyridoxal 5Ј-sulfatereconstituted OASS is inactive
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