Probing energy coupling in the yeast plasma membrane H+-ATPase with acetyl phosphate.

The energy-rich compound acetyl phosphate (ACP) was examined as a substrate for energy-linked reactions by the yeast plasma membrane H+-ATPase. The hydrolysis of ACP was sensitive to inhibition by vanadate with an IC50 approximately 1 microM, which is comparable to the level obtained in the presence of ATP. A Km of 8.29 +/- 0.65 mM for the hydrolysis of ACP was approximately 10-fold higher than that obtained for ATP, while Vmax values of 8.66 +/- 0.29 and 7.23 +/- 0.34 micromol Pi mg(-1) min(-1) were obtained with ATP and ACP, respectively. ACP formed a phosphorylated intermediate that was efficiently chased with hydroxylamine. Both ACP and ATP effectively protected the enzyme from trypsin-induced inactivation and formed identical tryptic digestion patterns, suggesting that ACP mimics the formation of conformational intermediates induced by ATP. However, unlike ATP, ACP was unable to drive proton transport by H+-ATPase. In addition, a pma1-S368F mutant enzyme that is highly insensitive to inhibition by vanadate in the presence of ATP was largely sensitive to vanadate in the presence of ACP. These results are interpreted in terms of a reverse, short-circuit pathway of the normal P-type ATPase kinetic pathway, in which the formation of E2P by-passes the E1P high-energy intermediate. In this pathway, ACP favors the formation of an E2P conformational state, which can interact with classical inhibitors like vanadate, but possesses insufficient free energy to drive proton transport by the H+-ATPase.