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首页> 外文期刊>Biochemistry >Exhaustive Mutagenesis of Six Secondary Active-Site Residues in Escherichia coli Chorismate Mutase Shows the Importance of Hydrophobic Side Chains and a Helix N-Capping Position for Stability and Catalysis
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Exhaustive Mutagenesis of Six Secondary Active-Site Residues in Escherichia coli Chorismate Mutase Shows the Importance of Hydrophobic Side Chains and a Helix N-Capping Position for Stability and Catalysis

机译：详尽的致突变性的大肠杆菌分支酸中的六个次要活性位点残留表明疏水性侧链和螺旋N封端位置对于稳定性和催化作用的重要性

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摘要

Secondary active-site residues in enzymes,including hydrophobic amino acids,may contribute to catalysis through critical interactions that position the reacting molecule,organize hydrogen-bonding residues,and define the electrostatic environment of the active site.To ascertain the tolerance of an important model enzyme to mutation of active-site residues that do not directly hydrogen bond with the reacting molecule,all 19 possible amino acid substitutions were investigated in six positions of the engineered chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase.The six secondary active-site residues were selected to clarify results of a previous test of computational enzyme design procedures.Five of the positions encode hydrophobic side chains in the wild-type enzyme,and one forms a helix N-capping interaction as well as a salt bridge with a catalytically essential residue.Each mutant was evaluated for its ability to complement an auxotrophic chorismate mutase deletion strain.Kinetic parameters and thermal stabilities were measured for variants with in vivo activity.Altogether,we find that the enzyme tolerated 34% of the 114 possible substitutions,with a few mutations leading to increases in the catalytic efficiency of the enzyme.The results show the importance of secondary amino acid residues in determining enzymatic activity,and they point to strengths and weaknesses in current computational enzyme design procedures.
机译：酶中的次要活性位点残基（包括疏水性氨基酸）可能通过关键相互作用来促进催化作用，该相互作用决定了反应分子的位置，组织了氢键残基并定义了活性位点的静电环境。确定重要模型的耐受性酶使不直接与反应分子发生氢键的活性位点残基突变，在大肠杆菌分支糖酸突变酶-苯甲酸酯脱水酶的工程化分支糖酸突变酶结构域的六个位置研究了所有19种可能的氨基酸取代。选择位点残基以澄清先前计算酶设计程序测试的结果。其中五个位置编码野生型酶中的疏水侧链，一个形成螺旋的N封端相互作用以及与催化必需残基评估每个突变体补充营养缺陷性绒毛的能力测量了具有体内活性的变异体的动力学参数和热稳定性。我们总共发现该酶耐受114种可能的取代中的34％，少数突变导致该酶催化效率的提高。结果表明仲氨基酸残基在确定酶活性中的重要性，并且指出了当前计算酶设计程序中的优点和缺点。

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