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
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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DOI：
10.1021/bi9911177
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发表时间：
1999-09-21
期刊：
影响因子：
2.9
通讯作者：
Whitman, CP
中科院分区：
文献类型：
--
作者：
Czerwinski, RM;Harris, TK;Whitman, CP
一般碱催化剂Pro-1在4-巴豆酸酯互变异构酶（4-OT）中的不寻常的低pK（a）值（pK（a）= 6.4）归因于活性位点处的低介电常数和阳离子残基Arg-11和Arg-39的接近性[Stivers，J.T.，Abeygunawardana，C.，Mildvan，A.美国，哈吉普尔G.，和惠特曼，C.（1996）Biochemistry 35，814-823]。此外，该研究中的pH速率曲线显示了一个未鉴定的质子化基团，该基团对催化至关重要，pK（a）为9.0。为了解决这些问题。活性位点Pro-1的pK（a）值和精氨酸残基的下限pK（a）值通过直接N-15 NMR pH滴定测定。Pro-1和必需酸组的pK（a）值独立于4-OT精氨酸突变体中4-OT动力学参数的pH-速率曲线确定，并与野生型进行比较。发现野生型4-OT和R11 A和R39 Q突变体中所有Arg N-共振的化学位移在4.9-9.7范围内与pH无关，表明没有精氨酸导致游离4-OT中酸性基团的动力学测定pK（a）为9.0。对于R11 A突变体，其中k（cat）/K-m降低了10倍（2.9），如直接N-15 NMR滴定（pK（a）= 6.3 +/-0.1）和k（cat）/K-m的pH依赖性（pK（a）= 6.4 +/-0.2）所揭示的，Pro-1的pK（a）与野生型酶的pK（a）相比没有显著改变。6.4 +/- 0.2）。R11 A突变体与二羧酸底物2-羟基粘康酸盐反应的k（cat）/K-m和k（cat）的pH-速率曲线均显示出驼峰，即，峰在与单羧酸底物2-羟基-2，4-异戊烯酸的反应中消失，表明与仅与二羧酸底物的双阴离子形式反应的野生型酶不同，R11 A突变体与6-COOH和6-COO-形式都反应，其中6-COOH形式的活性高12倍。在Arg-11突变为Ala时发生的底物的6-羧基的优选电离状态的这种逆转提供了Arg-11与底物的6-羧酸相互作用的强有力证据。在R39 Q突变体中，其中k（cat）/K-m降低了10（3）倍，动力学测定的Pro-1的pK（a）值为4.6 +/- 0.2，而Pro-1的电离显示负协同性，通过1D N-15 NMR测定的表观pK（a）为7.1 +/- 0.1。从希尔系数0.54可以看出，表观pK（a）值7.1可以最简单地由两个极限pK（a）值4.6和8.2的平均值得出。通过改变覆盖活性位点的β-发夹结构，Arg-39突变可能导致Pro-1的溶剂暴露增加，使其上限pK（a）值升高至8.2。在R39 A突变体中，Pro-1的动力学测定pK（a）也较低，为5.0 +/- 0.2，表明在R39 Q和R39 A突变体中，只有pK（a）值较低的位点具有动力学作用。对于完全活性的R61 A突变体，动力学测定的Pro-1的pK（a）（pK（a）= 6.5 +/-0.2）与野生型4-OT的pK（a）一致。和Arg-61残基，但结果主要来自低局部介电常数的位点。
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 N-15 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 N epsilon 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 N-15 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 N-15 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 beta-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.