The transition state of the phosphoryl-transfer reaction catalyzed by the lambda Ser/Thr protein phosphatase
The transition state of the phosphoryl-transfer reaction catalyzed by the lambda Ser/Thr protein phosphatase
复制标题
DOI：
10.1021/ja990667p
复制
发表时间：
1999-07-14
影响因子：
15
通讯作者：
Hengge, AC
中科院分区：
文献类型：
--
作者：
Hoff, RH;Mertz, P;Hengge, AC
使用重原子同位素效应和 pH 依赖性速率研究，研究了 Mn2+ 形式的天然噬菌体 lambda 磷酸酶和 H76N 突变体与底物磷酸对硝基苯酯的催化反应。底物中的动力学同位素效应是在非桥接氧原子 [(18)(V/K)（非桥）]、发生键断裂的桥接氧原子 [(18)(V/K)（桥）] 和硝基苯酚离去基团中的氮原子 [(15)(V/K)] 处测量的。在最佳 pH 值 7.8 下，天然酶的同位素效应对于 (18)(V/K)（桥）为 1.0133 +/- 0.0006，对于 (15)(V/K) 为 1.0006 +/- 0.0003，对于 (18)(V/K)（非桥）为 0.9976 +/- 0.0003。这些值在 6.0 至 9.0 的 pH 范围内的实验误差内保持恒定，并且对于当 Ca2+ 取代 Mn2+ 时产生的较慢的催化反应也没有变化。结果表明，P-O 键裂解的化学步骤是限速的，这是第一个已被证明是这种情况的金属磷酸酶。同位素效应与蛋白质酪氨酸磷酸酶反应测量的效应非常相似，表明这两个酶家族具有相似的解离过渡态。 H76N 突变体的 (18)(V/K)（桥）和 (15)(V/K) 同位素效应相对于天然酶在幅度上略有增加，但比离去基团带着完全负电荷离开时的预期值小得多。天然酶的 pH 与 k(cat) 曲线呈钟形，pK(a) 值为 7.7 +/- 0.3 和 8.6 +/- 0.4。在 pH 值范围 5.8-9.1 内，底物的 K-m 值随 pH 值增加约 70 倍。 H76N 突变体的 K-m 与高 pH 下天然酶观察到的 K-m 相似，并且在该 pH 范围内相对恒定。在 H76N 突变体反应中，pH 速率曲线的基本部分降低但并未消除。讨论了 His-76 的可能作用以及天然酶和突变体中催化过渡态的性质。
The catalytic reaction of the Mn2+ form of the native bacteriophage lambda phosphatase and the H76N mutant was studied with the substrate p-nitrophenyl phosphate using heavy atom isotope effects and pH-dependent rate studies. The kinetic isotope effects in the substrate were measured at the nonbridging oxygen atoms [(18)(V/K)(nonbridge)], at the bridging oxygen atom undergoing bond cleavage [(18)(V/K)(bridge)], and at the nitrogen atom in the nitrophenol leaving group [(15)(V/K)]. The isotope effects with native enzyme at the pH optimum of 7.8 were 1.0133 +/- 0.0006 for (18)(V/K)(bridge), 1.0006 +/- 0.0003 for (15)(V/K), and 0.9976 +/- 0.0003 for (18)(V/K)(nonbridge). These values were constant within experimental error across the pH range from 6.0 to 9.0 and were also unchanged for the slower catalytic reaction resulting when Ca2+ was substituted for Mn2+. The results indicate that the chemical step of P-O bond cleavage is rate-limiting, the first metallophosphatase for which this has been shown to be the case. The isotope effects are very similar to those measured for reactions of protein-tyrosine phosphatases, indicating that the two families of enzymes share similar dissociative transition states. The (18)(V/K)(bridge) and (15)(V/K) isotope effects for the H76N mutant were slightly increased in magnitude relative to the native enzyme but were much smaller than the values expected if the leaving group were departing with a full negative charge. The pH vs k(cat) profile for the native enzyme is bell-shaped with pK(a) values of 7.7 +/- 0.3 and 8.6 +/- 0.4. K-m values for substrate increased with pH approximately 70-fold across the pH range 5.8-9.1. The K-m for the H76N mutant was similar to that observed for native enzyme at high pH and was relatively constant across this pH range. The basic limb of the pH-rate profile is reduced but not abolished in the H76N mutant reaction. The results are discussed in terms of the possible role of His-76 and the nature of the transition state for catalysis in the native enzyme and mutant.