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

Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5′-triphosphate (ATP), orthophosphate (P i ), and pyruvate with adenosine 5′-monophosphate (AMP), pyrophosphate (PP i ), and phosphoenolpyruvate (PEP). The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent [ 32 P]-8-azidoadenosine 5′-triphosphate ([ 32 P]-8-azidoATP). Subtilisin cleavage of the [R-32 P]-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-32 P]-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. Comparison of the steady-state kinetic constants measured (at pH 6.8, 25°C) for wild-type PPDK (k cat ) 36 s -1 , AMP K m ) 7 µM, PP i K m ) 70 µM, PEP K m ) 27 µM) to those of R337L PPDK (k cat ) 2 s -1 , AMP K m ) 85 µM, PP i K m ) 3700 µM, PEP K m ) 6 µM) and G254I PPDK (k cat ) 0.1 s -1 , AMP K m ) 1300 µM, PP i K m ) 1200 µM, PEP K m ) 12 µM) indicated impaired catalysis of the nucleotide partial reaction (E‚ATP‚P i f E-PP‚AMP‚P i f E-P‚AMP‚PP i ) in these mutants. The single turnover reactions of [ 32 P]PEP to [ 32 P]E-P‚pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK. In contrast, the formation of [ 32 P]E-PP/[ 32 P]E-P in single turnover reactions of [ -32 P]ATP/P i was significantly inhibited. Finally, the location of the adenosine 5′-diphosphate binding site within the nucleotide binding domain of D-alanine-D-alanine ligase, a structural homologue of the PPDK N-terminal domain ) Proc. Natl. Acad. Sci. U.S.A. 93, 2652-2657 indicates, by analogy, the location of the nucleotide binding site in PPDK. Residues G254, R337, and E323 as well as the site of photoaffinity labeling are located within this region.