ADENINE-NUCLEOTIDE BINDING AT A NONCATALYTIC SITE OF MITOCHONDRIAL-F1-ATPASE ACCELERATES A MG2+-DEPENDENT AND ADP-DEPENDENT INACTIVATION DURING ATP HYDROLYSIS

The evidence is presented that the ADP- and Mg2+-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with k(inact) 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase, Acceleration of the MF1-ATPase inactivation by noncatalytic MgATP binding could reflect the promotion of P(i) release from the catalytic site to give the E.ADP complex required for inactivation by Mg2+. The interplay of Mg2+ and ADP inhibitory effects can account for previous suggestions of more than one apparent K(m) above micromolar ATP concentrations.