Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site
Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site
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DOI：
10.1111/j.1742-4658.2006.05266.x
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发表时间：
2006-06-01
期刊：
影响因子：
5.4
通讯作者：
Nakayama, Toru
中科院分区：
文献类型：
--
作者：
Okubo, Ayumi;Li, Mi;Nakayama, Toru
Kumamolisin-As 是一种具有枯草杆菌蛋白酶样折叠的酸性胶原酶。其活性位点包含独特的催化三联体 Ser278-Glu78-Asp82 和假定的过渡态稳定残基 Asp164。在本研究中，设计了突变体 D164N 和 E78H/DI64N，以便用枯草杆菌蛋白酶中相应位置的残基取代 kumamolisin-As 的部分催化机制。与野生型和 D164N 酶原不同，野生型和 D164N 酶原经过瞬时加工产生 37-kDa 成熟形式，表达的 E78H/DI64N 酶原作为前体的带切口和完整形式的平衡混合物存在。两种突变体成熟形式的 X 射线晶体结构表明，在每种突变体中，催化性 Ser278 与 Asn164 的侧链形成直接氢键。此外，双突变体的His78与Ser278和Asp82距离较远，催化三联体不再存在。与活性位点周围的这些结构改变一致，这些突变体仅表现出较低的催化活性（相对 k(cat) 在 pH 4.0 下，D164N 为 1.3%，E78H/DI64N 为 0.0001%）。 pH 依赖性动力学研究表明，单个 D164N 取代不会显着改变酶的 logk(cat) 与 pH 关系以及 log(k(cat)/K-m) 与 pH 关系曲线。相比之下，双突变导致 logk(cat) 与 pH 曲线的急剧转变，与 Ser278-His78 二联体和 Asn164 的催化作用一致，这也可能解释了观察到的 E78H/DI64N 前体的连接/切割平衡。这些结果证实了谷氨酸介导的催化三联体和氧阴离子稳定天冬氨酸残基对于酶的低pH肽酶活性的机械重要性。
Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/DI64N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature, forms, the expressed E78H/DI64N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/DI64N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/K-m) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/DI64N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.