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

• Published in: Biochemistry (Easton) 1996-07, Vol.35 (26), p.8544-8552
• Main Authors: McGuire, Marielena, Carroll, Lawrence J, Yankie, Linda, Thrall, Sara H, Dunaway-Mariano, Debra, Herzberg, Osnat, Jayaram, Beby, Haley, Boyd H
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - metabolism; Affinity Labels; Amino Acid Sequence; Binding Sites; Clostridium - enzymology; Clostridium symbiosum; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Pyruvate, Orthophosphate Dikinase - chemistry; Pyruvate, Orthophosphate Dikinase - genetics; Pyruvate, Orthophosphate Dikinase - metabolism; Zidovudine - analogs & derivatives
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi960275k

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 [α-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 α-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 α-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, PPi K m = 70 μM, PEP K m = 27 μM) to those of R337L PPDK (k cat = 2 s-1, AMP K m = 85 μM, PPi K m = 3700 μM, PEP K m = 6 μM) and G254I PPDK (k cat = 0.1 s-1, AMPKm = 1300 μM, PPi Km = 1200 μM, PEPKm = 12 μM) indicated impaired catalysis of the nucleotide partial reaction (E·ATP·Pi → E−PP·AMP·Pi → E−P·AMP·PPi) 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 [β-32P]ATP/Pi 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