Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida.
Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida.
复制标题
DOI：
10.1021/bi101761e
复制
发表时间：
2011-06-07
期刊：
影响因子：
2.9
通讯作者：
Ringe D
中科院分区：
文献类型：
--
作者：
Brodkin HR;Novak WR;Milne AC;D'Aquino JA;Karabacak NM;Goldberg IG;Agar JN;Payne MS;Petsko GA;Ondrechen MJ;Ringe D
活性位点可以被认为是残基层，其中直接与底物相互作用的残基也与第二壳中的残基相互作用，并且这些又与第三壳中的残基相互作用。第二层和第三层中的这些残基在保持第一壳催化残基的基本化学性质，特别是它们相对于底物结合口袋的空间排列以及它们的静电和动力学性质方面可能具有不同的作用。在何种程度上，这些远程残基参与催化，以及它们如何影响第一壳残基仍然是未知的。为了更好地理解第二和第三壳残基在催化中的作用，我们使用THEMATICS来鉴定来自恶臭假单胞菌的Co型腈水合酶（ppNHase）的第二和第三壳中可能对催化重要的残基。这些预测的残基中的五个，加上三个额外的，保守的残基，没有预测，已被保守突变，并研究其影响动力学和结构。所有这八个残基与活性位点金属离子或结合底物没有直接接触。这些结果表明，三个预测的第二壳残基，α-Asp 164，β-Glu 56和β-His 147，和一个预测的第三壳残基β-His 71，对酶的催化效率有显着影响。其中一个预测的残基α-Glu 168和三个未预测的残基α-Arg 170、α-Tyr 171和β-Tyr 215对酶的催化效率没有任何显著影响。
Active sites may be regarded as layers of residues, whereby the residues that interact directly with substrate also interact with residues in a second shell, and these in turn interact with residues in a third shell. These residues in the second and third layers may have distinct roles in maintaining the essential chemical properties of the first-shell catalytic residues, particularly their spatial arrangement relative to the substrate binding pocket, and their electrostatic and dynamic properties. The extent to which these remote residues participate in catalysis and precisely how they affect first-shell residues remains unexplored. In order to better understand the roles of second- and third-shell residues in catalysis, we used THEMATICS to identify residues in the second- and third-shells of the Co-type nitrile hydratase from Pseudomonas putida (ppNHase) that may be important for catalysis. Five of these predicted residues, plus three additional, conserved residues that were not predicted, have been conservatively mutated, and their effects studied both kinetically and structurally. All of these eight residues have no direct contact with the active site metal ion or bound substrate. These results demonstrate that three of the predicted second-shell residues, α-Asp164, β-Glu56, and β-His147, and one predicted third-shell residue β-His71, have significant effects on the catalytic efficiency of the enzyme. One of the predicted residues, α-Glu168, and the three residues not predicted, α-Arg170, α-Tyr171, and β-Tyr215, do not show any significant effects on the catalytic efficiency of the enzyme.
登录
查看更多内容
影响因子：
5.8
作者：
Glaser, F;Pupko, T;Ben-Tal, N
通讯作者：
Ben-Tal, N
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
15
作者：
Arakawa, Takatoshi;Kawano, Yoshiaki;Odaka, Masafumi
通讯作者：
Odaka, Masafumi
DOI：
10.1073/pnas.82.23.7840
发表时间：
1985-01-01
影响因子：
11.1
作者：
LEATHERBARROW, RJ;FERSHT, AR;WINTER, G
通讯作者：
WINTER, G
影响因子：
2.9
作者：
Bas, Delphine C.;Rogers, David M.;Jensen, Jan H.
通讯作者：
Jensen, Jan H.