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

Published

July 1, 1999

Author(s)

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

Abstract

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 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 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 ¹⁵N NMR pH titrations. The pK_avalues 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 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_aof Pro-1 was not significantly altered from that of wild type enzyme (pK_a= 6.4 + 0.2) as revealed by both direct ¹⁵N 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 10³, the kinetically determined pKd_a value for Pro-1 was 4.6 plus or minus} 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK_a 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 pK_a value of 7.1 could result most simply from the averaging of two limiting pK_a 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 pK_avalue to 8.2. In the R39A mutant, the kinetically determined pK_a of Pro-1 was also low, 5.0 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK_avalues were kinetically operative. With the fully active R61A mutant, the kinetically determined pK_aof Pro-1 (pK_a = 6.5 0.2) agreed with that of wild type 4-OT. It is concluded that the unusually low pK_aof 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.

Citation

Biochemistry

Volume

38

Pub Type

Journals

Keywords

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

Citation

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_a Values of Active Site Residues and on the pH Dependence of Catalysis, Biochemistry (Accessed April 25, 2024)