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.
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