Determination of the nucleotide binding site within Clostridium symbiosum pyruvate phosphate dikinase by photoaffinity labeling, site-directed mutagenesis, and structural analysis

作者：McGuire M; Carroll LJ; Yankie L; Thrall SH; DunawayMariano D; Herzberg O; Jayaram B; Haley BH

来源：Biochemistry, 1996, 35(26): 8544-8552.

DOI：10.1021/bi960275k

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 (PPi), and phosphoenolpyruvate (PEP). The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent [P-32]-8-azidoadenosine 5'-triphosphate ([P-32]-8-azidoATP). Subtilisin cleavage of the [alpha-P-32]-8-azidoATP-photolabeled PPDK into domain-sized fragments, prior to SDS-PAGE analysis, allowed us to identify two sites of modification: one between residues I 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 [gamma-P-32]-8-azidoATP-photolabeled PPDK were generated by treating the denatured protein with trypsin or alpha-chymotrypsin, A pair of overlapping radiolabeled peptide fragments were separated from the two digests, DMQDMEFTLEEGK (positions 318-330 in trypsin-treated PPDK) and RDMQDMEFTIEEGKL (positions 317-331 in alpha-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 degrees C) for wild-type PPDK (k(cat) = 36 s(-1), K-AMP(m) = 7 mu M, K-PPi(m) = 70 mu M, K-PEP(m) = 27 mu M) to those of R337L PPDK (k(cat) = 2 s(-1), K-AMP(m) = 85 mu M, K-PPi(m) = 3700 mu M, K-PEP(m) = 6 mu M) and G254I PPDK (k(cat) = 0.1 s(-1), K-AMP(m) = 1300 mu M, K-PPi(m) = 1200 mu M, K-PEP(m) = 12 mu M) indicated impaired catalysis of the nucleotide partial reaction (E . ATP . P-i --> E-PP . AMP . P-i-->E-P . AMP . PPi) in these mutants. The single turnover reactions of [P-32]PEP to [P-32]E-P . pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK. In contrast, the formation of [P-32]E-PP/[P-32]E-P in single turnover reactions of [beta(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 [Herzberg, O. (1996) 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.

• 出版日期1996-7-2