Synthesis of ATP during oxidative phosphorylation occurs on the F1F0-ATP synthase enzyme and accounts for the bulk of ATP synthesis in living cells; the goal of this research is to understand the structure and mechanism of action of F1F0-ATP synthase in as much molecular detail as possible. The E. coli enzyme will be studied because of its several advantages e.g. it is readily amenable to molecular biology/genetic manipulation, it may be dissociated into individual native subunits and reassociated, and it may be obtained in high yield. Specific goals are (1) to study structure and function of the catalytic sites by mutagenesis, reversion analysis, and enzymological techniques (2) to study the remarkable positive catalytic cooperativity which is a crucial mechanistic feature of this enzyme by mutagenesis and reversion analysis (3) to construct chimerical genes from alpha and betasubunit genes, to express and purify the chimerical proteins, and to study their nucleotide binding properties in order to distinguish determinant features of the adenine-specific, non-catalytic alpha-sites from those of the catalytic #-sites (4) to characterize intrinsic catalytic steps of single-site catalysis in terms of pH and solvent effects in normal and mutant enzymes (5) to study energy coupling between the FD proton-pathway and F1 catalytic sites by mutagenesis of beta and &-subunits, reversion analysis, and development of assays of Lpinduced changes in nucleotide-binding g affinity and catalytic site environment. ATP-driven pumps are very widely distributed in nature, and are involved in many disease states; work to be done here will consequently have broad impact in biology and medicine.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM025349-20
Application #
2518894
Study Section
Special Emphasis Panel (NSS)
Project Start
1978-09-01
Project End
2001-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
20
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Ahmad, Zulfiqar; Senior, Alan E (2006) Inhibition of the ATPase activity of Escherichia coli ATP synthase by magnesium fluoride. FEBS Lett 580:517-20
Ahmad, Zulfiqar; Senior, Alan E (2005) Modulation of charge in the phosphate binding site of Escherichia coli ATP synthase. J Biol Chem 280:27981-9
Ahmad, Zulfiqar; Senior, Alan E (2005) Identification of phosphate binding residues of Escherichia coli ATP synthase. J Bioenerg Biomembr 37:437-40
Wilkens, Stephan; Borchardt, Dan; Weber, Joachim et al. (2005) Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy. Biochemistry 44:11786-94
Ahmad, Zulfiqar; Senior, Alan E (2005) Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding. FEBS Lett 579:523-8
Weber, Joachim; Senior, Alan E (2004) Fluorescent probes applied to catalytic cooperativity in ATP synthase. Methods Enzymol 380:132-52
Weber, Joachim; Wilke-Mounts, Susan; Nadanaciva, Sashi et al. (2004) Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase. J Biol Chem 279:11253-8
Weber, Joachim; Muharemagic, Alma; Wilke-Mounts, Susan et al. (2004) Analysis of sequence determinants of F1Fo-ATP synthase in the N-terminal region of alpha subunit for binding of delta subunit. J Biol Chem 279:25673-9
Senior, Alan E; Weber, Joachim (2004) Happy motoring with ATP synthase. Nat Struct Mol Biol 11:110-2
Ahmad, Zulfiqar; Senior, Alan E (2004) Mutagenesis of residue betaArg-246 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F1-ATPase. J Biol Chem 279:31505-13

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