Separate site catalysis by pyruvate phosphate dikinase as revealed by deletion mutants.
Separate site catalysis by pyruvate phosphate dikinase as revealed by deletion mutants.
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缺失突变体揭示了丙酮酸磷酸二激酶的单独位点催化作用。
DOI：
10.1021/bi00007a013
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Martin,BM
中科院分区：
文献类型：
--
作者：
Xu,Y;McGuire,M;Dunaway-Mariano,D;Martin,BM
1994年10月28日接收的修订版Mandarin pt ®摘要：以前的研究表明，丙酮酸磷酸二激酶（PPDK），一种催化腺苷5 '-三磷酸（ATP）、正磷酸（Pi）和丙酮酸与腺苷5'-单磷酸（AMP）、焦磷酸（PPi）和磷酸烯醇丙酮酸（PEP）相互转化的酶，由25、13、18、和35 kDa结构域[卡罗尔，L. J.，徐，Y.，萨尔，S. H、马丁，B。M.的情况，& Dunaway-Mariano，D.（1994）Biochemistry 33，1134]，催化组氨酸（其介导磷酰基从ATP转移到Pi和丙酮酸）位于18 kDa结构域上，而25和13 kDa结构域似乎分别含有ATP结合位点，35 kDa结构域似乎含有丙酮酸结合位点。本研究的目的是检查功能的相互依赖性的推定ATP和ATP结合域。通过使用重组DNA技术产生两种截短形式的PPDK。发现35 kDa（C-末端）缺失突变体催化E+ ATP+ P i = EP+ AMP+ PP i部分反应，但不催化EP+丙酮酸E+ PEP部分反应。发现25 kDa（N-末端）缺失突变体催化EP+丙酮酸盐/酯+ PEP部分反应，但不催化E+ ATP+ P;* EP+ AMP+ PP;部分反应。两种突变体都不催化完整的ATP+ Pi+丙酮酸 * AMP+ PPi+ PEP反应。丙酮酸磷酸二激酶（PPDK）1在某些微生物和C4植物中催化ATP、丙酮酸和Pi与AMP、PEP和PPi的相互转化（Wood等人，1977年）。该酶的一个新特征是使用组氨酸残基从ATP中提取Pp-Py焦磷酸单位，并依次将yP磷酰基和f-P磷酰基分别传递给Pi和丙酮酸（方案1）。该反应以三个化学步骤进行，包括焦磷酸酶中间体和磷酸酶中间体的形成和反应（Wood等人，1977;卡罗尔等人，1989; Thrall等人，1993年）。为方便起见，将前两个步骤组合以形成所谓的核苷酸部分反应（E+ ATP+ P;
Revised Manuscript Received October 28, 1994® abstract: Previous studies had indicated that pyruvate phosphate dikinase (PPDK), an enzyme which catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PPi), and phosphoenolpyruvate (PEP), is made up of 25, 13, 18, and 35 kDa domains [Carroll, L. J., Xu, Y., Thrall, S. H., Martin, B. M., & Dunaway-Mariano, D.(1994) Biochemistry 33, 1134], The catalytic histidine (which mediates the phosphoryl group transfers from ATP to Pi and pyruvate) is located on the 18 kDa domain while the 25 and 13 kDa domains appear to contain the ATP binding site and the 35 kDa domain appears to contain the pyruvate binding site, respectively. The goal of this investigation was to examine functional interdependency of the putative ATP and pyruvatebinding domains. Two truncated forms of PPDK were created by using recombinant DNA techniques. The 35 kDa (C-terminal) deletion mutant was found to catalyzethe E+ ATP+ P;= EP+ AMP+ PPj partial reaction but notthe EP+ pyruvate E+ PEP partial reaction. The 25 kDa (N-terminal) deletion mutant was found to catalyze the EP+ pyruvate^ E+ PEP partial reaction but not the E+ ATP+ P;*» EP+ AMP+ PPj partial reaction. Neither mutant catalyzes the full ATP+ Pi+ pyruvate*** AMP+ PPi+ PEP reaction. These results are interpreted to mean that the ATP and pyruvate binding domains in PPDK are functionally independent, thus providing evidence for separate active sites for catalysis of the two partial reactions.Pyruvate phosphate dikinase (PPDK) 1 catalyzes the interconversion of ATP, pyruvate, and Pi with AMP, PEP, and PPi in certain microorganisms and in C4 plants (Wood et al., 1977). A novelfeature of this enzyme is the use of a histidine residue to abstractthe Pp—Py pyrophosphate unit from ATP and sequentiallydeliver the yP phosphoryl and f}-P phosphoryl group to Pi and pyruvate, respectively (Scheme 1). The reaction takes place in three chemical steps involving the formation and reaction of a pyrophosphorylenzyme intermediate and a phosphorylenzyme intermediate (Wood et al., 1977; Carroll et al., 1989; Thrall et al., 1993). For convenience the first two steps are combined to form what is referred to as the nucleotidepartial reaction (E+ ATP+ P;—