The influence of pH on the interaction of inhibitors with triosephosphate isomerase and determination of the pKa of the active-site carboxyl group.
Ionization effects on the binding of the potential transition state analogues 2-phosphoglycolate and 2-phosphoglycolohydroxamate appear to be attributable to the changing state of ionization of the ligands themselves, therefore it is unnecessary to postulate the additional involvement of an ionizing residue at the active site of triosephosphate isomerase to explain the influence of changing pH on Ki in the neutral range. The binding of the competitive inhibitor inorganic sulfate is insensitive to changing pH in the neutral range. 3-Chloroacetol sulfate, synthesized as an active-site-specific reagent for triosephosphate isomerase, is used to provide an indication of the pKa of the essential carboxyl group of this enzyme. Previously described active-site-specific reagents for the isomerase were phosphate esters, and their changing state of ionization (accompanied by possible changes in their affinity for the active site) may have complicated earlier attempts to determine the pKa of the essential carboxyl group from the pH dependence of the rate of inactivation. Being a strong monoprotic acid, chloroacetol sulfate is better suited to the determination of the pKa of the carboxyl group. Chloroacetol sulfate inactivates triosephosphate isomerase by the selective esterification of the same carboxyl group as that which is esterified by the phosphate esters described earlier. From the pH dependence of the rate of inactivation of yeast triosephosphate isomerase, the apparent pKa of the active-site carboxyl group is estimated as 3.9 +/- 0.1.
D000818:Animals; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011485:Protein Binding; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*
D006902:Hydroxymercuribenzoates; D004228:Dithionitrobenzoic Acid; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase