ALLOSTERIC REGULATION OF PROTON TRANSLOCATION BY A VACUOLAR ADENOSINE-TRIPHOSPHATASE

The kinetics of nucleoside-triphosphate-dependent proton translocation by a vacuolar-type adenosine-triphosphatase have been studied, using the enzyme from bovine chromaffin-granule membranes, purified and reco nstituted into proteoliposomes. The reaction was followed by recording the quenching of the fluorescence of the permeant weak base 9-amino-6 -chloro-2-methoxyacridine I fluorescence data were collected and store d in digital form, and the initial reaction rates estimated by linear regression. In the absence of nucleoside diphosphate, the dependence o f initial rates of proton translocation on substrate concentration wer e fitted well by the Michaelis-Menten equation, as were the kinetics o f ATP hydrolysis. ADP and other nucleoside diphosphates were potent in hibitors of the ATPase, effecting a reduction in the maximum velocity of the reaction, and producing sigmoid substrate-saturation curves whi ch could be fitted by the empirical Hill equation, the Hill coefficien t approaching 2 at high inhibitor concentrations, Data sets containing initial-rate estimates were collected over a wide range of independen tly varied concentrations of substrate and inhibitor and were modeled, using rate equations derived from several different models based on t he concerted-transition model of allosteric inhibition proposed by Mon od, Wyman and Changeux. These equations were fitted to the data by wei ghted non-lineal regression, using an iterative computer program to ob tain the best estimates of kinetic parameters. One model consistently fitted all of the data sets better than all the others, and this model was based on the following assumptions: that the ATPase exists in two conformational states, R and T; that only the R state is catalyticall y active; that each state contains three kinetically equivalent cataly tic sites, and one regulatory site; that nucleoside triphosphates bind only to the catalytic sites, and that nucleoside diphosphates bind bo th to the catalytic sites and to the regulatory site. The optimized va lues of the kinetic parameters indicate that in the absence of nucleos ide diphosphate, the enzyme is almost completely in the R state; that nucleoside triphosphates bind more tightly to the R than to the T stat e; that binding of nucleoside diphosphates to the regulatory site is v ery tight, but occurs only in the T state; and that competitive bindin g of nucleoside diphosphates at the catalytic sites is stronger in the T state than in the R state. Experiments conducted with varying total magnesium concentrations indicated that the magnesium complexes of nu cleoside diphosphates are much stronger inhibitors than the free nucle otides, and that free nucleoside triphosphates are weakly inhibitory, probably competing with the magnesium complexes for binding at the cat alytic sites. The results of these experiments indicate that the effec ts of nucleoside diphosphates, particularly ABP, occur at concentratio n ranges that are likely to be physiologically significant, and they m ake predictions about ligand-induced conformation changes in the ATPas e that can be tested by other means.