The F 1 F 0 proton-translocating ATPase/synthase is the primary generator of ATP in most organisms growing aerobically. Kinetic assays of ATP synthesis have been conducted using enzymes from mitochondria and chloroplasts. However, limited data on ATP synthesis by the model Escherichia coli enzyme are available, mostly because of the lack of an efficient and reproducible assay. We have developed an optimized assay and have collected synthase kinetic data over a substrate concentration range of 2 orders of magnitude for both ADP and P i from the synthase enzyme of E. coli. Negative and positive cooperativity of substrate binding and positive catalytic cooperativity were all observed. ATP synthesis displayed biphasic kinetics for ADP indicating that 1) the enzyme is capable of catalyzing efficient ATP synthesis when only two of three catalytic sites are occupied by ADP; and 2) occupation of the third site further activates the rate of catalysis.The F-type proton-translocating ATPase/synthases are a family of enzymes primarily responsible for the oxidative phosphorylation of ADP to form ATP (1-3). These membrane-bound enzymes are found in the inner membranes of mitochondria, the thylakoid membranes of chloroplasts, and the inner membranes of bacteria. The synthase uses energy from an electrochemical gradient of protons generated by a membrane-bound electron transport chain to drive the binding and catalysis of ADP and P i and the release of ATP. The actual synthesis (or hydrolysis in the reverse direction) reaction of the triphosphate nucleotide occurs in one of three catalytic sites under tightly bound conditions (the "tight" site or site 1) with a K eq ϳ 1; thus, the energy provided by the proton gradient is used primarily to alter the binding of the substrates and products, and hence the "binding change" mechanism.Certain aspects of the enzymes are generally accepted. In the direction of hydrolysis, given an enzyme with three empty catalytic sites, MgATP binds to the first site with very high affinity (nanomolar range). Once bound, this nucleotide undergoes oxygen exchange with medium water (a process called unisite catalysis) as the nucleotide undergoes rapid cycles of hydrolysis and resynthesis. Release of ADP is very slow (4, 5).As the concentration of ATP is increased and approaches the K d values for the remaining two sites, the rate of product release increases, a phenomenon called catalytic cooperativity (6). The affinity of the second site for MgATP (micromolar range) is lower than the first, and the affinity of the third site is lower still (range, 10 -100 M). Thus, the binding of nucleotide exhibits negative cooperativity.There has been controversy recently regarding the necessity of substrate binding to subsequent sites for the activation of rapid catalysis. On one hand, it has been argued and demonstrated that some enzymes, particularly those from mitochondria and chloroplasts, display bisite activation, i.e. the binding of substrate(s) at the second site is necessary and sufficient to accelerate the ...