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不等弯孢菌含钒氯过氧化物酶在酿酒酵母中的异源表达及活性位点残基组氨酸(496)、赖氨酸(353)、精氨酸(360)和精氨酸(490)的定点诱变。

Heterologous expression of the vanadium-containing chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His(496), Lys(353), Arg(360), and Arg(490).

作者信息

Hemrika W, Renirie R, Macedo-Ribeiro S, Messerschmidt A, Wever R

机构信息

E. C. Slater Institute, Faculty of Chemistry, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.

出版信息

J Biol Chem. 1999 Aug 20;274(34):23820-7. doi: 10.1074/jbc.274.34.23820.

Abstract

The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.

摘要

来自不等弯孢菌的含钒氯过氧化物酶在酿酒酵母中实现了高水平的异源表达。对重组酶的表征表明,其行为与天然氯过氧化物酶非常相似。对四个高度保守的活性位点残基进行定点诱变,以研究它们在催化中的作用。当钒酸盐结合残基His(496)突变为丙氨酸时,突变酶失去了与钒酸盐共价结合的能力,导致酶失活。钒酸盐氧原子上的负电荷通过与残基Arg(360)、Arg(490)和Lys(353)形成的氢键得到补偿。当这些残基突变为丙氨酸时,突变酶失去了有效氧化氯离子的能力,但仍可作为溴过氧化物酶发挥作用。提出了卤过氧化物酶催化的一般机制,该机制还将突变体的动力学性质与钒酸盐结合位点的电荷和氢键网络联系起来。

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