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

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• 作者：Robert M. Czerwinski ; Thomas K. Harris ; William H. Johnson ; Jr. ; Patricia M. Legler ; James T. Stivers ; Albert S. Mildvan ; and Christian P. Whitman
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：September 21, 1999
• 年：1999
• 卷：38
• 期：38
• 页码：12358 - 12366
• 全文大小：132K
• 年卷期：v.38,no.38(September 21, 1999)
• ISSN：1520-4995

文摘

The unusually low pK_a value of the general base catalyst Pro-1 (pK_a = 6.4) in 4-oxalocrotonatetautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximityof 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 thatstudy showed an unidentified protonated group essential for catalysis with a pK_a of 9.0. To address theseissues, the pK_a values of the active site Pro-1 and lower limit pK_a values of arginine residues weredetermined by direct ¹⁵N NMR pH titrations. The pK_a values of Pro-1 and of the essential acid groupwere determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutantsof 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Ns/gifchars/epsilon.gif" BORDER=0 > resonances inwild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK_a of 9.0 for an acidicgroup 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 ofPro-1 was not significantly altered from that of the wild-type enzyme (pK_a = 6.4 ± 0.2) as revealed byboth 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 dicarboxylatesubstrate, 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 dicarboxylicsubstrate, the R11A mutant reacts with both the 6-COOH and 6-COO^- forms, with the 6-COOH formbeing 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of thesubstrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts withthe 6-carboxylate of the substrate. In the R39Q mutant, where k_cat/K_m was reduced by a factor of 10³, thekinetically determined pK_a value for Pro-1 was 4.6 ± 0.2, while the ionization of Pro-1 showed negativecooperativity with an apparent pK_a of 7.1 ± 0.1 determined by 1D ¹⁵N NMR. From the Hill coefficientof 0.54, it can be shown that the apparent pK_a value of 7.1 could result most simply from the averagingof two limiting pK_a values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-hairpinwhich covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upperlimit 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 kineticallyoperative. 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 littlecontribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues butresults primarily from a site of low local dielectric constant.