YEAST MITOCHONDRIAL F1-ATPASE - EFFECTS OF METAL-IONS

The effects of a series of metal ions (M) on the velocity (V) of the r eaction catalyzed by the yeast mitochondrial F1-ATPase were investigat ed in N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and Imida zole buffers (pH 7.45), with total concentrations of ATP ranging from 2 to 8 mM. The biphasic metal ion activation/inhibition effects, obser ved with a constant total amount of ATP, were found to be consistent w ith a simple empirical kinetic equation, V-1/V = 1 + K-1/[M] + [M]/K-2 , where V-1, K-1, and K-2 are empirical parameters and [M] the concent rations of metal ions not complexed with the ATP. Three alternative ki netic equations, involving the concentrations of either ATP or its met al chelate, consistent with this empirical equation, were shown to be invalid since changing the total amount of ATP did not affect the empi rical reaction parameters V-1, K-1, and K-2. The fact that changing th e total ATP concentration failed to change the empirical reaction para meters suggests that the inhibition by an excess of ATP, which is obse rved with a constant total metal ion concentration, is due to chelatio n of free metal ions that are required for the ATP hydrolysis. When th e total amount of added ATP was in excess of that of added magnesium i ons it was found that calcium, strontium, and aluminum ions, which lib erate free magnesium from the Mg-ATP chelate by complexing the ATP, al l activated. These activations apparently confirm the fact that the AT Pase requires a free magnesium ion as a cofactor for the hydrolysis of ATP. (C) 1994 Academic Press, Inc.