6-PYRUVOYL TETRAHYDROPTERIN SYNTHASE, AN ENZYME WITH A NOVEL TYPE OF ACTIVE-SITE INVOLVING BOTH ZINC-BINDING AND AN INTERSUBUNIT CATALYTIC TRIAD MOTIF - SITE-DIRECTED MUTAGENESIS OF THE PROPOSED ACTIVE-CENTER, CHARACTERIZATION OF THE METAL-BINDING SITE AND MODELING OF SUBSTRATE-BINDING
6-PYRUVOYL TETRAHYDROPTERIN SYNTHASE, AN ENZYME WITH A NOVEL TYPE OF ACTIVE-SITE INVOLVING BOTH ZINC-BINDING AND AN INTERSUBUNIT CATALYTIC TRIAD MOTIF - SITE-DIRECTED MUTAGENESIS OF THE PROPOSED ACTIVE-CENTER, CHARACTERIZATION OF THE METAL-BINDING SITE AND MODELING OF SUBSTRATE-BINDING
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DOI：
10.1006/jmbi.1995.0558
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发表时间：
1995-10-20
影响因子：
5.6
通讯作者：
NAR, H
中科院分区：
文献类型：
--
作者：
BURGISSER, DM;THONY, B;NAR, H
6-丙酮酰四氢蝶呤合成酶(PTPS)是参与四氢生物蝶呤生物合成的酶，是多种芳香族氨基酸单加氧酶和一氧化氮合酶的辅因子。最近对PTPS的晶体结构进行了解析，结果表明它是由一个三聚体组成的同源六聚体。在每个亚基中发现了由23、48和50三个组氨酸残基形成的过渡金属结合部位。金属分析和apo-PTPs的重组表明，锌(II)是结合的过渡金属，对酶活性起决定作用。这三个组氨酸残基的定点突变都会导致金属结合和酶活性的完全丧失。位于金属结合部位附近的三个残基，Cys42，His89和Glu133，先前被认为参与了催化反应。我们改变了这些残基，发现突变的C42A完全失去了酶活性。两个突变体H89N和E133Q的酶活力分别为4.3%和1.3%，但底物的K-M值与野生型PTPs相似。基于这些结果，我们提出了底物与活性中心匹配的模型，并描述了一个新的由氨基酸残基Cys42、His89和Asp88组成的亚基间催化三联体。与大多数催化酰胺键或酯键的催化三元化合物不同，PTPS活性中心的催化三元化合物似乎参与了底物侧链碳的去质子化。我们的模型还提出了锌(II)作为两个底物侧链羟基的配位以及Glu133作为立体专一性质子服务器的参与。(C)1995年学术出版社有限公司
6-Pyruvoyl tetrahydropterin synthase (PTPS) is an enzyme involved in tetrahydrobiopterin biosynthesis, the cofactor for several aromatic amino acid monooxygenases and the nitric oxide synthases. The crystal structure of PTPS was recently solved and showed a homohexameric enzyme composed of a dimer of trimers. A transition metal binding site formed by the three histidine residues 23, 48 and 50 was found in each subunit. We showed by metal analysis and reconstitution of apo-PTPS that Zn(II) was the bound transition metal and responsible for the enzymatic activity. Site-directed mutagenesis of each of these three histidine residues resulted in a complete loss of metal binding and enzymatic activity. The three residues, Cys42, His89 and Glu133, located close to the metal binding site, were previously postulated to be involved in the catalytic reaction. We altered these residues and found a complete loss of enzymatic activity for the mutant C42A. The two mutants, H89N and E133Q, showed 4.3% and 1.3% enzymatic activity, respectively, but had similar K-M values for the substrate as compared to wild-type PTPS. Based on these results we propose a model of the substrate fitted into the active site and we describe a novel intersubunit catalytic triad motif composed of the amino acid residues Cys42, His89 and Asp88. Different from most other catalytic triads that catalyse the hydrolysis of an amide or ester bond, the catalytic triad in the active site of PTPS seems to be involved in the deprotonation of the substrate's side-chain carbons. Our model also proposes Zn(II) as the coordination site for the two substrate side-chain hydroxy groups as well as the involvement of Glu133 as putative stereospecific proton server. (C) 1995 Academic Press Limited