McGuire M, Huang K, Kapadia G, Herzberg O, Dunaway-Mariano D
Department of Chemistry and Biochemistry, University of Maryland, College Park 27042, USA.
Biochemistry. 1998 Sep 29;37(39):13463-74. doi: 10.1021/bi980920i.
Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and pyruvate with AMP, PPi, and PEP in three partial reactions: (1) E + ATP --> E.ATP --> E-PP.AMP, (2) E-PP.AMP + Pi --> E-PP.AMP.Pi --> E-P.AMP.PPi, and (3) E-P + pyruvate --> E-P.pyruvate --> E.PEP. The Clostridium symbiosum PPDK structure consists of N-terminal, central, and C-terminal domains. The N-terminal and central domains catalyze partial reactions 1 and 2 whereas the C-terminal and central domains catalyze partial reaction 3. The goal of the present work is to determine where on the N-terminal domain catalysis of partial reactions 1 and 2 occurs and, in particular, where the Pi binding site is located. Computer modeling studies implicated Arg337 as a key residue for Pi binding. This role was tested by site-directed mutagenesis. The R337A PPDK was shown to be impaired in catalysis of the forward (kcat 300-fold lower) and reverse (kcat 30-fold lower) full reactions. Time courses for the single turnover reactions were measured to show that catalysis of partial reaction 1 is 5-fold slower in the mutant, catalysis of the second partial reaction is 140-fold slower in the mutant, and catalysis of the third partial reaction is unaffected. With the exception of the mutation site, the crystal structure of the R337A PPDK closely resembles the structure of the wild-type protein. Thus, the altered kinetic properties observed for this mutant are attributed solely to the elimination of the interaction between substrate and the guanidinium group of the Arg337 side chain. On the basis of these findings we propose that the Pi binding site is located within the crevice of the PPDK N-terminal domain, at a site that is flanked by the ATP beta-P and the Mg2+ cofactor.
丙酮酸磷酸双激酶（PPDK）在三个部分反应中催化ATP、磷酸（Pi）和丙酮酸与AMP、焦磷酸（PPi）和磷酸烯醇式丙酮酸（PEP）的相互转化：（1）E + ATP → E·ATP → E-PP·AMP，（2）E-PP·AMP + Pi → E-PP·AMP·Pi → E-P·AMP·PPi，以及（3）E-P + 丙酮酸 → E-P·丙酮酸 → E-PEP。共生梭菌PPDK结构由N端、中央和C端结构域组成。N端和中央结构域催化部分反应1和2，而C端和中央结构域催化部分反应3。本研究的目的是确定部分反应1和2的催化在N端结构域的何处发生，特别是Pi结合位点位于何处。计算机建模研究表明精氨酸337（Arg337）是Pi结合的关键残基。通过定点诱变对这一作用进行了测试。结果表明，R337A PPDK在正向（催化常数kcat降低300倍）和反向（催化常数kcat降低30倍）全反应的催化中受损。测量了单周转反应的时间进程，结果表明，突变体中部分反应1的催化速度慢5倍，第二个部分反应的催化速度慢140倍，第三个部分反应的催化不受影响。除了突变位点外，R337A PPDK的晶体结构与野生型蛋白的结构非常相似。因此，观察到的该突变体动力学性质的改变完全归因于底物与Arg337侧链胍基之间相互作用的消除。基于这些发现，我们提出Pi结合位点位于PPDK N端结构域的裂隙内，该位点两侧是ATP的β-磷酸和Mg2+辅因子。
