Analysis of nucleotide binding by a vacuolar proton-translocating adenosine triphosphatase.

Abstract

The vacuolar-type proton-translocatine adenosine triphosphatase from bovine adrenal secretory granules (chromaffin granules) was purified and reconstituted into proteoliposomes. The binding of nucleotides to the enzyme was studied by quantifying their effects on the rate of inactivation by N-ethylmaleimide (MalNEt) of ATP-dependent proton translocation, and by direct measurement of the binding of [3H]MgADP. The results of these experiments are consistent with a model of the enzyme that had been developed as a result of kinetic experiments, the features of which are that the enzyme exists in two states, each containing three nucleotide-binding sites on catalytic subunits, and that nucleoside diphosphates regulate the enzyme by binding with high affinity to a single site in the inactive T state of the enzyme. Under the conditions of the experiments, MalNEt inactivated the ATPase in a pseudo-first order reaction. Rate constants of inactivation were reduced in the presence of MgADP, MgIDP and free ADP; the kinetics of protection suggested that the two conformational states of the enzyme were inactivated at different rates and also confirmed the existence of two different types of binding site for MgADP. Low nucleotide concentrations afforded partial protection from MalNEt; this was ascribed to binding of nucleotide to the regulatory site causing a shift in the conformational equilibrium towards the T state, which was more slowly inactivated than the unliganded R state of the enzyme. At higher nucleotide concentrations, binding at the catalytic site afforded complete protection from MalNEt. Protection by MgADP[S] and magnesium 2'- and 3'-O-[4-benzoylbenzoyl]adenosine 5'-triphosphate showed simpler kinetics but was also consistent with previously reported kinetic results. Analysis of subunit labelling with [3H]MalNEt showed that the three 72-kDa (catalytic) subunits were alkylated by MalNEt with similar rate constants, consistent with a symmetrical arrangement of the catalytic subunits, in contrast to the situation in F-type ATPases. Analysis of the binding of [3H]MgADP also confirmed the results of kinetic experiments. MgADP was shown to bind to the enzyme with an apparent dissociation constant of about 66 nM; assuming that the nucleotide binds only to the T-state, the true dissociation constant is < 1 nM. Using Blue Native polyacrylamide gel electrophoresis to separate the holo-ATPase from the membrane sector, the stoichiometry of binding was calculated to be 0.6 mol/mol enzyme, confirming the existence of a single regulatory site for MgADP. However, binding of MgADP to the enzyme was much slower than could be accounted for by the measured dissociation constants, suggesting that it is rate limited by a step such as a protein conformational change. Treatment designed to remove endogenous nucleotide had no effect on the rate or extent of binding of MgADP.