The pH dependence of kinetic parameters using natural and alternative reactants was determined in order to obtain information on the chemical mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium. A general mechanism is proposed for OASS in which OAS binds with its α-amine unprotonated to carry out a nucleophilic attack on C4’ of the protonated Schiff base and with the acetyl carbonyl hydrogen-bonded to a protonated enzyme group (or a water molecule), which aids in the β-elimination of acetate. The enzyme lysine that was in Schiff base linkage with the active site pyridoxal 5'-phosphate deprotonates the α-carbon in the β-elimination reaction, and a proton is likely released with the acetate product. Sulfide likely binds as HS- to undergo nucleophilic attack on the -aminoacrylate intermediate, followed by protonation of the α-carbon by the enzyme lysine. In OASS-A, HS- is hydrogen-bonded to the enzyme group that assists in the β-elimination of acetate, but this is not the case for OASS-B. The pH independent equilibrium constant for the first half-reaction of OASS-A is 1.6 x 10-3, while the second half-reaction is practically irreversible.
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