Characterization of the 2-[(R)-2-hydroxypropylthio]ethanesulfonate dehydrogenase from Xanthobacter strain Py2: product inhibition, pH dependence of kinetic parameters, site-directed mutagenesis, rapid equilibrium inhibition, and chemical modification.
Although the short-chain dehydrogenase/reductase (SDR) superfamily contains a very large number of members defined in annotated databases and by biochemical and structural studies, very few SDR enzymes have been identified that have a homologous partner catalyzing the same reaction but with an opposite stereospecificity. In the present study we have cloned and expressed one of these enzymes, the 2-[(R)-2-hydroxypropylthio]ethanesulfonate (R-HPC) dehydrogenase, that is part of the coenzyme M-dependent pathway of alkene and epoxide metabolism in Xanthobacter strain Py2. Investigation of the kinetic mechanism using product inhibition suggested that a compulsory-ordered ternary complex mechanism was followed. The pH dependence of k(cat)/K(m) indicated the presence of a single ionizable residue of catalytic importance (pK(a) = 6.9) that was proposed to be Y155 of the catalytic triad. Amino acid substitutions of the putative catalytic triad residues produced inactive enzymes (S142C, Y155F, Y155E, and K159A) or enzyme with a greatly decreased activity (S142A). Inhibitors were investigated as probes of the molecular features of R-HPC that contribute to substrate binding. 2-[(S)-2-Hydroxypropylthio]ethanesulfonate (S-HPC) and 2-(2-methyl-2-hydroxypropylthio)ethanesulfonate were found to be competitive inhibitors of R-HPC with K(ic) values close to the K(m) for R-HPC. The arginine-specific modifiers 2,3-butanedione and phenylglyoxal were found to be inactivators, and inactivation could be protected against by the addition of R-HPC. 2,3-Butanedione was found to reduce enzyme activity with R-HPC as a substrate much more dramatically than with substrates that lacked a sulfonate moiety [e.g., 2-propanol, (R)-2-pentanol, and (R)-2-heptanol]. Amino acid analyses of enzyme modified by 2,3-butanedione in the presence and absence of S-HPC suggested protection of a single arginine residue. On the basis of these results, we propose that one or more active site arginines play a key role in substrate binding via an ionic interaction with the sulfonate moiety of R-HPC.
Clark
Daniel D
DD
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Ensign
Scott A
SA
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D017931:DNA Primers; D003931:Diacetyl / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D020584:Xanthobacter / Q000201:enzymology* / Q000254:growth & development
D000596:Amino Acids; D017931:DNA Primers; D000429:Alcohol Oxidoreductases; C450599:2-hydroxypropylthioethanesulfonate dehydrogenase, Xanthobacter; D003931:Diacetyl