The catalytic site for ATP synthesis by mitochondrial and bacterial oxidative phosphorylation is on a membrane-bound enzyme, F1, that can be readily prepared in a soluble form. The structure, mechanism, and regulation of F1 is the subject of this proposal. Rate constants for several elementary steps in catalysis by F1 will be determine by direct measurements employing rapid kinetic techniques, oxygen exchange reactions, isotope trap procedures, and Sephadex centrifuge column binding assays. The effects that enzyme modification and purification of monomeric catalytic subunit have on the apparent strong cooperative interactions between multiple catalytic sites will be studied. The results may also provide information regarding the number of functional sites and their location within or between subunits. The adenine nucleotide sites on F1 show asymmetric behavior. Experiments are designed to determine whether this is due to ligand-induced site-site cooperativity or to permanent structural asymmetry. Some of the nucleotide sites appear to have an regulatory role. We will prepare F1 with different known compositions of adenine nucleotide at these sites and measure transient and steady-state kinetic properties of soluble and membrane-bound enzyme. It has been proposed that F1 has separate catalytic sites for ATP synthesis and ATP hydrolysis. We will examine possible alternative explanations for several of the observations cited in support of this idea. Energized release of 125I-labeled mitochondrial inhibitor protein will be used as a measure of the fraction of F1 that participates in ATP synthesis of following modification by """"""""uni-directional"""""""" reagents. Also, measurements of rate constants for GTP and ITP cleavage at a single site may explain the failure of these substrates to drive energy-requiring membrane processes. F1 binds two anionic substrates and has essential lysine and arginine residues that may participate in change interactions at the catalytic site. We have designed and synthesized a new affinity probe for lysine at adenine nucleotide sites. Using this reagent and phenylglyoxal we will attempt to modify, isolate, and sequence peptides containing essential residues.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023152-08
Application #
3271512
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1976-05-01
Project End
1989-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
8
Fiscal Year
1985
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
Bulygin, Vladimir V; Milgrom, Yakov M (2007) Studies of nucleotide binding to the catalytic sites of Escherichia coli betaY331W-F1-ATPase using fluorescence quenching. Proc Natl Acad Sci U S A 104:4327-31
Milgrom, Yakov M; Cross, Richard L (2005) Rapid hydrolysis of ATP by mitochondrial F1-ATPase correlates with the filling of the second of three catalytic sites. Proc Natl Acad Sci U S A 102:13831-6
Bulygin, Vladimir V; Duncan, Thomas M; Cross, Richard L (2004) Rotor/Stator interactions of the epsilon subunit in Escherichia coli ATP synthase and implications for enzyme regulation. J Biol Chem 279:35616-21
Cross, Richard L; Muller, Volker (2004) The evolution of A-, F-, and V-type ATP synthases and ATPases: reversals in function and changes in the H+/ATP coupling ratio. FEBS Lett 576:1-4
Hutcheon, M L; Duncan, T M; Ngai, H et al. (2001) Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the F(O) sector of Escherichia coli ATP synthase. Proc Natl Acad Sci U S A 98:8519-24
Cross, R L (2000) The rotary binding change mechanism of ATP synthases. Biochim Biophys Acta 1458:270-5
Milgrom, Y M; Murataliev, M B; Boyer, P D (1998) Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase. Biochem J 330 ( Pt 2):1037-43
Bulygin, V V; Duncan, T M; Cross, R L (1998) Rotation of the epsilon subunit during catalysis by Escherichia coli FOF1-ATP synthase. J Biol Chem 273:31765-9
Milgrom, Y M; Cross, R L (1997) Nucleotide-depleted beef heart F1-ATPase exhibits strong positive catalytic cooperativity. J Biol Chem 272:32211-4
Zhou, Y; Duncan, T M; Cross, R L (1997) Subunit rotation in Escherichia coli FoF1-ATP synthase during oxidative phosphorylation. Proc Natl Acad Sci U S A 94:10583-7

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