The long term goal of the project is to understand the mechanism of oxidative phosphorylation with regard to the molecular mechanism of ATp formation on the mitochondrial ATPase (F1) and the molecular mechanism of coupling between the energy site in the mitochondrial membrane and the ATP synthesizing apparatus. This project considers that during oxidative phosphorylation ATP is formed in catalytic sites on F1 from bound ADP and Pi with virtually zero change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP from high affinity catalytic sites. The project will examine the reaction mechanism of the ATPase in soluble and membrane-bound forms. It will determine whether the action of the catalytic sites is simultaneous or sequential, it will study changes in the enzyme subsequent to substrate binding that would be indicative of conformational change and it will study pH-dependent changes in the equilibrium constants of the reaction mechanism, particularly changes in product release, as an approach to the nature of the interactions of the ATPase with the coupling mechanism. A study of the catalytic site cooperativity and of reversible Pi and ATP-dependent activation of the enzyme are expected to provide further insight into the reacton mechanism of the enzyme. The methodological approaches to be employed include quenched flow measurements for determining equilibrium constants in the reaction mechanism, stop flow techniques to search for intermediates in the initial steps of ATP hydrolysis and the centrifuge column.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021737-24
Application #
3270642
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1988-09-15
Project End
1989-12-31
Budget Start
1989-01-01
Budget End
1989-12-31
Support Year
24
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Upstate Medical University
Department
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Knowles, A F; Penefsky, H S (1997) Reconstitution of beef heart mitochondrial F0F1 in reverse phase evaporation vesicles. Biochim Biophys Acta 1329:311-20
Souid, A K; Penefsky, H S (1995) Energetics of ATP dissociation from the mitochondrial ATPase during oxidative phosphorylation. J Biol Chem 270:9074-82
Martins, I S; Penefsky, H S (1994) Covalent modification of catalytic sites on membrane-bound beef heart mitochondrial ATPase by 2-azido-adenine nucleotides. Eur J Biochem 224:1057-65
Xiao, R; Penefsky, H S (1994) Unisite catalysis and the delta subunit of F1-ATPase in Escherichia coli. J Biol Chem 269:19232-7
Souid, A K; Penefsky, H S (1994) Mechanism of ATP synthesis by mitochondrial ATP synthase from beef heart. J Bioenerg Biomembr 26:627-30
Ziegler, M; Xiao, R; Penefsky, H S (1994) Close proximity of Cys64 and Cys140 in the delta subunit of Escherichia coli F1-ATPase. J Biol Chem 269:4233-9
Ziegler, M; Penefsky, H S (1993) The adenine nucleotide translocase modulates oligomycin-induced quenching of pyranine fluorescence in submitochondrial particles. J Biol Chem 268:25320-8
Freedman, J C; Novak, T S; Penefsky, H S et al. (1992) Quantitative analysis of oxonol V fluorescence in submitochondrial particles. Ann N Y Acad Sci 671:493-6
Penefsky, H S; Cross, R L (1991) Structure and mechanism of FoF1-type ATP synthases and ATPases. Adv Enzymol Relat Areas Mol Biol 64:173-214
Penefsky, H S (1988) Rate of chase-promoted hydrolysis of ATP in the high affinity catalytic site of beef heart mitochondrial ATPase. J Biol Chem 263:6020-2

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