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

Robert M. Czerwinski, Thomas K. Harris, William H. Johnson, Patricia M. Legler, James T. Stivers, Albert S. Mildvan, Christian P. Whitman

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46 Scopus citations

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 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(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 Nε 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 102.9, 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 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 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 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(a) value 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(a) values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK(a) of Pro-1 (pK(a) = 6.5 ± 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK(a) of 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.

Original languageEnglish (US)
Pages (from-to)12358-12366
Number of pages9
JournalBiochemistry
Volume38
Issue number38
DOIs
StatePublished - Sep 21 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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