One major, ultimate goal of the research proposed here is to elucidate the molecular mechanism of ATP hydrolysis catalyzed by the F1-ATPases. The F1-ATPases are the catalytic components of the ATP synthases present in energy transducing membranes. Therefore, elucidation of the molecular mechanism of the hydrolytic reaction catalyzed by the soluble F1-ATPase will have great bearing on the elucidation of the molecular mechanism of ATP synthesis catalyzed by the intact ATP synthases. To achieve these goals, chemical modification studies are being conducted in parallel on the F1-ATPases from bovine heart mitochondria (MF1), the plasma membrane of E. coli (EF1), and the plasma membrane of the thermophilic bacterium, PS3 (TF1). Site specific radioactive or fluorescent reagents which alter the activities of these enzymes are being used to identify the amino acid side chains which participate in catalytic and/or cooperative events. Using these techniques we have identified five amino acid residues, all residing in the Beta subunit, which are essential for the hydrolytic reaction catalyzed by the F1-ATPases. These are: Tyr-368, which is modified by 5'-p-fluorosulfonyl-benzoyladenosine (FSBA); Tyr-311, which reacts with 7-chloro-4-nitrobenzofurazan (Nbf-Cl); Lys-162, which is labeled when the Nbf-group migrates to it from Tyr-311; Glu-188, which reacts with dicyclohexylcarbodiimide (DCCD) in TF1; and Glu-199, which reacts with DCCD in MF1 and EF1. For only one of these residues, Tyr-368, is there unequivocal evidence that it resides at the catalytic site. The immediate goals of the research proposed on the F1-ATPases are to elucidate the functional roles of the essential residues which have been identified and to use other site specific reagents to identify additional amino acid side chains which participate directly in catalysis or in the propagation of signals between interacting adenine nucleotide binding sites. The other major goal of the research described in this proposal is to provide a thorough structural characterization of the polypeptide or polypeptides which comprise the lysosomal H+-ATPase. An immediate goal of the research on the lysosomal H+-ATPase is to obtain and sequence labeled peptides obtained after inactivating the enzyme with radioactive FSBA, DCCD, or N-ethylmaleimide or with quinacrine mustard which is fluorescent.
