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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



R M. Czerwinski, T K. Harris, W H. Johnson, P M. Legler, J T. Stivers, A S. Mildvan, C P. Whitman


The unusually low pKa value of the general base catalyst Pro-1 (pKa = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and to 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 pKa^of 9.0. To address these issues, the pKa values of the active site Pro-1 and lower limit pKa values of arginine residues were determined by direct 15N NMR pH titrations. The pKavalues 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 Nξ 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 to 9.7, indicating that no arginine is responsible for the kinetically determined pKa of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where kcat/Km was reduced by a factor of 102.9, the pKaof Pro-1 was not significantly altered from that of wild type enzyme (pKa= 6.4 + 0.2) as revealed by both direct 15N NMR titration (pKa = 6.3 + 0.1) and the pH dependence of kcat/Km (pKa= 6.4 + 0.2). The pH-rate profiles of both kcat/Km and kcat 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 kcat/Km was reduced by a factor of 103, the kinetically determined pKda value for Pro-1 was 4.6 plus or minus} 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pKa of 7.1 plus or minus} 0.1 determined by 1D 15N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pKa value of 7.1 could result most simply from the averaging of two limiting pKa values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the Β-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pKavalue to 8.2. In the R39A mutant, the kinetically determined pKa of Pro-1 was also low, 5.0 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pKavalues were kinetically operative. With the fully active R61A mutant, the kinetically determined pKaof Pro-1 (pKa = 6.5 0.2) agreed with that of wild type 4-OT. It is concluded that the unusually low pKaof Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant.


negative cooperativity, NMR, pH-rate profile, proton transfer


Czerwinski, R. , Harris, T. , Johnson, W. , Legler, P. , Stivers, J. , Mildvan, A. and Whitman, C. (1999), Effects of Mutations of the Active Site Arginine Residues in-4-Oxalocrotonate Tautomerase on the pK<sub>a</sub> Values of Active Site Residues and on the pH Dependence of Catalysis, Biochemistry (Accessed April 19, 2024)
Created June 30, 1999, Updated October 12, 2021