Effects of Mutations of the Active Site Arginine Residues in 4-Oxalocrotonate Tautomerase on the pKa Values of Active Site Residues and on the pH Dependence of Catalysis

The unusually low pK(a) value of the general base catalyst Pro-1 (pK(a) = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. In addition, the pH-rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK(a) of 9.0. To address these issues, the pK(a) values of the active site Pro-1 and lower limit pK(a) values of arginine residues were determined by direct (15)N NMR pH titrations. The pK(a) values of Pro-1 and of the essential acid group were determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nepsilon resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK(a) of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k(cat)/K(m) was reduced by a factor of 10(2.9), the pK(a) of Pro-1 was not significantly altered from that of the wild-type enzyme (pK(a) = 6.4 +/- 0.2) as revealed by both direct (15)N NMR titration (pK(a) = 6.3 +/- 0.1) and the pH dependence of k(cat)/K(m) (pK(a) = 6.4 +/- 0.2). The pH-rate profiles of both k(cat)/K(m) and k(cat) for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO(-) forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k(cat)/K(m) was reduced by a factor of 10(3), the kinetically determined pK(a) value for Pro-1 was 4.6 +/- 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK(a) of 7.1 +/- 0.1 determined by 1D