Determination of the Nucleotide Binding Site within Clostridium symbiosum Pyruvate Phosphate Dikinase by Photoaffinity Labeling, Site-Directed Mutagenesis, and Structural Analysis†

ABSTRACT: Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5′-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5′-monophosphate (AMP), pyrophosphate (PPi), and phosphoenolpyruvate (PEP). The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent [32P]-8-azidoadenosine 5′-triphosphate ([32P]-8-azidoATP). Subtilisin cleavage of the [R-32P]-8-azidoATP-photolabeled PPDK into domain-sized fragments, prior to SDS-PAGE analysis, allowed us to identify two sites of modification: one between residues 1 and 226 and the other between residues 227 and 334. Saturation of the ATP binding site with adenylyl imidodiphosphate afforded protection against photolabeling. Next, small peptide fragments of [γ-32P]-8-azidoATP-photolabeled PPDK were generated by treating the denatured protein with trypsin or R-chymotrypsin. A pair of overlapping radiolabeled peptide fragments were separated from the two digests, DMQDMEFTIEEGK (positions 318-330 in trypsin-treated PPDK) and RDMQDMEFTIEEGKL (positions 317-331 in R-chymotrypsin-treated PPDK), thus locating one of the positions of covalent modification. Next, catalysis by site-directed mutants generated by amino acid replacement of invariant residues of the PPDK N-terminal domain was tested. K163L, D168A, D170A, D175A, K177L, and G248I PPDK mutants retained substantial catalytic activity while G254I, R337L, and E323L PPDK mutants were inhibited