This project proposes new approaches to an old problem, namely how adenosine triphosphate (ATP) is made using energy from oxidations and light. ATP serves to drive many biological processes, and an active human turns over about a body weight of ATP each day. How this is accomplished is basic to an understanding of how optimum health can be maintained. One principal objective will be to make critical tests of a proposed mechanism for ATP synthesis. We will use heart and liver mitochondria, chloropast thylakoids, and E. coli membranes to explore the following suggested features of this binding change mechanism: conformational changes driven by the electrochemical potential of the coupling membrane serve to promote competent binding of Pi and ADP and the release of ATP from catalytic sites of the ATP synthase; three catalytic sites on the multisubunit synthase participate in coordinated alternation; and during ATP synthesis there is a rotational change in position of catalytic subunits relative to noncatalytic core subunits occurs. A second major objective will be to explore how the energy-requiring conformational changes are driven: indirectly by the electrochemical potential or directly by oxidation-reduction enzymes. A new concept for energy transmission to the synthase from oxidation-reduction enzymes is presented for experimental testing. Procedures proposed include selected kinetic evaluations; measurements of amounts and properties of bound intermediates; measurement of phosphate-oxygen exchanges and of the 18O-species formed; application of present and the development of new specific cross-linking agents; chemical derivatization with various agents and inhibitors; effects of catalysis on the chemical reactivity and on the physical properties of bound agents; energy transfer measurements between intrinsic and added probes; the effects of selected mutants of the E. coli enzyme on catalytic parameters; the role of subunits as probed by deletion and recombination experiments; and the possible crystallization of the catalytic Beta-subunit for X-ray studies. Plans also call for assembly and publication of the methodology for 18O analysis in phosphates that we have accummulated over the past several years.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM011094-26
Application #
3268233
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1975-05-01
Project End
1990-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
26
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Murataliev, M B; Boyer, P D (1994) Interaction of mitochondrial F1-ATPase with trinitrophenyl derivatives of ATP and ADP. Participation of third catalytic site and role of Mg2+ in enzyme inactivation. J Biol Chem 269:15431-9
Boyer, P D (1993) The binding change mechanism for ATP synthase--some probabilities and possibilities. Biochim Biophys Acta 1140:215-50
Zhou, J M; Boyer, P D (1993) Evidence that energization of the chloroplast ATP synthase favors ATP formation at the tight binding catalytic site and increases the affinity for ADP at another catalytic site. J Biol Chem 268:1531-8
Zhou, J M; Boyer, P D (1992) MgADP and free Pi as the substrates and the Mg2+ requirement for photophosphorylation. Biochemistry 31:3166-71
Murataliev, M B; Boyer, P D (1992) The mechanism of stimulation of MgATPase activity of chloroplast F1-ATPase by non-catalytic adenine-nucleotide binding. Acceleration of the ATP-dependent release of inhibitory ADP from a catalytic site. Eur J Biochem 209:681-7
Murataliev, M B; Milgrom, Y M; Boyer, P D (1991) Characteristics of the combination of inhibitory Mg2+ and azide with the F1 ATPase from chloroplasts. Biochemistry 30:8305-10
Milgrom, Y M (1991) When beef-heart mitochondrial F1-ATPase is inhibited by inhibitor protein a nucleotide is trapped in one of the catalytic sites. Eur J Biochem 200:789-95
Berkich, D A; Williams, G D; Masiakos, P T et al. (1991) Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis. J Biol Chem 266:123-9
Milgrom, Y M; Ehler, L L; Boyer, P D (1991) The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase. J Biol Chem 266:11551-8
Milgrom, Y M; Boyer, P D (1990) The ADP that binds tightly to nucleotide-depleted mitochondrial F1-ATPase and inhibits catalysis is bound at a catalytic site. Biochim Biophys Acta 1020:43-8

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