LOCATION OF THE PHOSPHATE-BINDING SITE WITHIN CLOSTRIDIUM-SYMBIOSUM PYRUVATE PHOSPHATE DIKINASE

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of AT P, P-i, and pyruvate with AMP, PPi, and PEP in three partial reactions : (1) E + ATP --> E.ATP --> E-PP.AMP, (2) E-PP. AMP + P-i --> E-PP.AMP .P-i --> E-P.AMP.PPi, and (3) E-P + pyruvate --> E-P.pyruvate --> E.PE P. The Clostridium symbiosum PPDK structure consists of N-terminal, ce ntral, and C-terminal domains. The N-terminal and central domains cata lyze partial reactions 1 and 2 whereas the C-terminal and central doma ins catalyze partial reaction 3. The goal of the present work is to de termine where on the N-terminal domain catalysis of partial reactions 1 and 2 occurs and, in particular, where the P-i binding site is locat ed. Computer modeling studies implicated Arg337 as a key residue for P -i binding. This role was tested by site-directed mutagenesis. The R33 7A PPDK was shown to be impaired in catalysis of the forward (k(cat) 3 00-fold lower) and reverse (k(cat) 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, cataly sis of the second partial reaction is 140-fold slower in the mutant, a nd catalysis of the third partial reaction is unaffected. With the exc eption of the mutation site, the crystal structure of the R337A PPDK c losely resembles the structure of the wild-type protein. Thus, the alt ered kinetic properties observed for this mutant are attributed solely to the elimination of the interaction between substrate and the guani dinium group of the Arg337 side chain. On the basis of these findings we propose that the P-i 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.