title: 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 (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 [32P]-8-azidoadenosine 5'-triphosphate ([32P]-8-azidoATP). Subtilisin cleavage of the [alpha-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 [gamma-32P]- 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, DMQDMEFTIEEGK (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 (kcat = 36 s-1, AMPK(m) = 7 microM, PP(i)K(m) = 70 microM, PEPK(m) = 27 microM) to those of R337L PPDK (kcat = 2 s-1, AMPK(m) = 85 microM, PP(i)K(m) = 3700 microM, PEPK(m) = 6 microM) and G254I PPDK (kcat = 0.1 s-1, AMPK(m) = 1300 microM, PP(i)K(m) = 1200 microM, PEPK(m) = 12 microM) indicated impaired catalysis of the nucleotide partial reaction (E.ATP.P(i) --> E-PP.AMP.P(i) --> E-P.AMP.PP(i) in these mutants. The single turnover reactions of [32P]PEP to [32P]E-P.pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK. In contrast, the formation of [32P]E-PP/[32P]E-P in single turnover reactions of [beta-32P]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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
authors.forename: M
authors.initials: M
authors.lastname: McGuire
authors.forename: L J
authors.initials: LJ
authors.lastname: Carroll
authors.forename: L
authors.initials: L
authors.lastname: Yankie
authors.forename: S H
authors.initials: SH
authors.lastname: Thrall
authors.forename: D
authors.initials: D
authors.lastname: Dunaway-Mariano
authors.forename: O
authors.initials: O
authors.lastname: Herzberg
authors.forename: B
authors.initials: B
authors.lastname: Jayaram
authors.forename: B H
authors.initials: BH
authors.lastname: Haley
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D003013:Clostridium / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011772:Pyruvate, Orthophosphate Dikinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015215:Zidovudine / Q000031:analogs & derivatives
chemical_list: D000227:Adenine Nucleotides; D000345:Affinity Labels; D015215:Zidovudine; D011772:Pyruvate, Orthophosphate Dikinase