Apparent equivalence of the active-site glutamyl residue and the essential group with pK a 6.0 in triosephosphate isomerase

Abstract

Summary An anomaly has existed with respect to the catalytic group of triosephosphate isomerase that effects proton transfer: Based on kinetic studies the group has a pKa of 6.0, whereas based on studies with affinity labels the group is a glutamyl carboxyl with pKa of 3.9. To ascertain if these two pKas represent two different active-site residues, we selected yeast isomerase for an evaluation of the pH-dependencies of Vmax and KM, since this enzyme, in contrast to muscle isomerase used in earlier kinetic work, is sufficiently stable to permit studies at the low pHs necessary to discern a catalytic group with pKa 3.9. From pH 4.2 to 7.4 profiles of both Vmax vs. pH and Vmax/KM vs. pH exhibit single ionizations that correspond to groups with pKas of 4.6 and 5.9, respectively. Since only one group can be detected in the Vmax profile, we conclude that it represents the essential glutamyl carboxyl and that the acidity of this carboxyl is lowered to different extents in the muscle and yeast enzymes upon substrate binding. We propose that the group with pKa 5.9 seen in the Vmax/KM profile is the secondary ionization of the substrate's phosphate group.