| Abstract
| - O-Acetylserine sulfhydrylase is a pyridoxal 5‘-phosphate-dependent enzyme that catalyzesthe final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of theβ-acetoxy group of O-acetyl-l-serine by a thiol to give l-cysteine. Two isozymes are found in Salmonellatyphimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobicconditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 Å and exhibits overalla fold very similar to that of the A-isozyme. The main difference between the two isozymes is the morehydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutralresidues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and iswithin hydrogen-bonding distance to Y286, while C280 is located about 3.4 Å from the pyridine nitrogen(N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than theA-isozyme and an ∼10-fold lower Km for O-acetyl-l-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the α-aminoacrylateintermediate at any concentration of O-acetyl-l-serine. The Kd values for formation of the external Schiffbase with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0−7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observethe pKa for the α-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effectresulting from titration of a group on the enzyme with a pKa in the vicinity of the α-amine's pKa. The pHdependence of the first-order rate constant for decay of the α-aminoacrylate intermediate to give pyruvateand ammonia gives a pKa of about 9 for the active site lysine (K41), a pH unit higher than that of theA-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa forK41, and the preference for the neutral species at high pH compared to the A-isozyme can be explainedby titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserinesulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emissionspectra of the internal aldimine in the absence and presence of the product acetate and of the externalaldimine with l-serine. The two isozymes show a different equilibrium distribution of the enolimine andketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B.The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In thepresence of acetate, which occupies the α-carboxylate subsite, the equilibrium between tautomers is shiftedtoward the ketoenamine tautomer, as a result of a conformational change affecting the structure of theactive site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylaseB-isozyme a higher degree of conformational flexibility linked to catalysis.
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