title: Comparative Mg(2+)-dependent sequential covalent binding stoichiometries of 3'-O-(4-benzoyl)benzoyl adenosine 5'-diphosphate of MF1, TF1, and the alpha 3 beta 3 core complex of TF1. The binding change motif is independent of the F1 gamma delta epsilon subunits.
abstract: Three F1 preparations, the beef heart (MF1) and thermophilic bacterium (TF1) holoenzymes, and the alpha 3 beta 3 "core" complex of TF1 reconstituted from individually expressed alpha and beta subunits, were compared as to their kinetic and binding stoichiometric responses to covalent photoaffinity labeling with BzATP and BzADP (+/- Mg2+). Each enzyme displayed an enhanced pseudo-first order rate of photoinhibition and one-third of the sites covalent binding to a catalytic site for full inhibition, plus, but not minus Mg2+. Titration of near stoichiometric [MgBzADP]/[F1] ratios during photolysis disclosed two sequential covalent binding patterns for each enzyme; a high affinity binding corresponding to unistoichiometric covalent association concomitant with enzyme inhibition, followed by a low affinity multisite-saturating covalent association. Thus, in the absence of the structural asymmetry inducing gamma delta epsilon subunits of the holoenzyme, the sequential binding of nucleotide at putative catalytic sites on the alpha 3 beta 3 complex of any F1 appears sufficient to effect binding affinity changes. With MF1, final covalent saturation of BzADP-accessible sites was achieved with 2 mol of BzADP/mol of enzyme, but with TF1 or its alpha 3 beta 3 complex, saturation required 3 mol of BzADP/mol of enzyme. Such differential final labeling stoichiometries could arise because of the endogenous presence of 1 nucleotide already bound to one of the 3 potential catalytic sites on normally prepared MF1, whereas TF1, possessing no endogenous nucleotide, has 3 vacant BzADP-accessible sites. Kinetics measurements revealed that regardless of the incremental extent of inhibition of the TF1 holoenzyme by BzADP during photolysis, the two higher apparent Km values (approximately 1.5 x 10(-4) and approximately 10(-3) M, respectively) of the progressively inactivated incubation are unchanged relative to fully unmodified enzyme. As reported for BzATP (or BzADP) and MF1 (Ackerman, S.H., Grubmeyer, C., and Coleman, P.S. (1987) J. Biol. Chem. 262, 13765-13772), this supports the fact that the photocovalent inhibition of F1 is a one-hit one-kill phenomenon. Isoelectric focusing gels revealed that [3H]BzADP covalently modifies both TF1 and MF1 exclusively on the beta subunit, whether or not Mg2+ is present. A single 19-residue [3H]BzADP-labeled peptide was resolved from a tryptic digest of MF1, and this peptide corresponded with the one believed to contain at least a portion of the beta subunit catalytic site domain (i.e. beta Ala-338----beta Arg-356).
authors.affiliation: Department of Biology, New York University, New York 10003.
authors.forename: P
authors.initials: P
authors.lastname: Aloise
authors.forename: Y
authors.initials: Y
authors.lastname: Kagawa
authors.forename: P S
authors.initials: PS
authors.lastname: Coleman
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014357:Trypsin
chemical_list: D000345:Affinity Labels; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C045291:3'-O-(4-benzoyl)benzoyladenosine diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; D008274:Magnesium