ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 344, No. 2, August 15, pp. 309–315, 1997 Article No. BB970213 Probing Energy Coupling in the Yeast Plasma Membrane H / -ATPase with Acetyl Phosphate Genfu Wang and David S. Perlin 1 Public Health Research Institute, 455 First Avenue, New York, New York 10016 Received February 7, 1997, and in revised form May 19, 1997 late intracellular pH (1). The H / -ATPase belongs to the The energy-rich compound acetyl phosphate (ACP) P-type class of ion-translocating ATPases that includes was examined as a substrate for energy-linked reac- the Na / ,K / -ATPase of animal cell plasma membranes, tions by the yeast plasma membrane H / -ATPase. The the Ca 2/ -ATPase of sarcoplasmic reticulum and red hydrolysis of ACP was sensitive to inhibition by vana- blood cells, and the H / ,K / -ATPase of gastric mem- date with an IC 50 Ç1 mM, which is comparable to the branes. All P-type enzymes couple ATP hydrolysis to level obtained in the presence of ATP. A K m of 8.29 { the translocation of ions, share a number of important 0.65 mM for the hydrolysis of ACP was approximately catalytic features including the formation of an acyl 10-fold higher than that obtained for ATP, while V max phosphate intermediate, and are strongly inhibited by values of 8.66 { 0.29 and 7.23 { 0.34 mmol P i mg 01 min 01 vanadate. The primary structures believed to comprise were obtained with ATP and ACP, respectively. ACP the ATP binding and phosphorylation domains are formed a phosphorylated intermediate that was effi- highly conserved, and it is expected that these enzymes ciently chased with hydroxylamine. Both ACP and share a common fundamental mechanism for energy ATP effectively protected the enzyme from trypsin-in- coupling (2 – 4). duced inactivation and formed identical tryptic diges- Simple energy-rich phosphate donors such as acetyl tion patterns, suggesting that ACP mimics the forma- phosphate (ACP) 2 are known to react with P-type en- tion of conformational intermediates induced by ATP. zymes to form phosphorylated intermediates that are However, unlike ATP, ACP was unable to drive proton rapidly hydrolyzed, and there has been considerable transport by H / -ATPase. In addition, a pma1-S368F interest in ACP as a probe of the catalytic reaction cycle mutant enzyme that is highly insensitive to inhibition for this class of enzymes (5–10). ACP has been shown by vanadate in the presence of ATP was largely sensi- to elicit different functional responses that could help tive to vanadate in the presence of ACP. These results distinguish potential differences in energy-coupling are interpreted in terms of a reverse, short-circuit mechanisms between the various P-type enzymes. For pathway of the normal P-type ATPase kinetic path- way, in which the formation of E 2 P by-passes the E 1 P example, acetyl phosphate supports calcium transport high-energy intermediate. In this pathway, ACP favors by the Ca 2/ -ATPase from sarcoplasmic reticulum (9) the formation of an E 2 P conformational state, which and Na / and K / transport by the Na / ,K / -ATPase (11), can interact with classical inhibitors like vanadate, but it does not support the transport of H / or K / by the but possesses insufficient free energy to drive proton gastric H / ,K / -ATPase (5). The functional differences transport by the H / -ATPase.  1997 Academic Press elicited by acetyl phosphate are also observed as differ- Key Words: H / -ATPase; proton transport; coupling; ences in intermediates of the reaction cycle. It has been acetyl phosphate. shown for the Ca 2/ -ATPase that, with the exception of the phosphorylation step, the rate constants for the reaction of acetyl phosphate in the presence of calcium are consistent with those for the reaction with ATP at The plasma membrane H / -ATPase of Saccharomyces subactivating concentrations (7). In addition, the phos- cerevisiae is an electrogenic proton pump that main- phorylation of the H / ,K / -ATPase from ACP appears to tains electrochemical proton gradients and helps regu- result in the formation of a low-energy E 2 P enzyme 1 To whom correspondence should be addressed. Fax: (212) 578- 2 Abbreviations used: ACP, acetyl phosphate; TCA, trichloroacetic acid; DMSO, dimethyl sulfoxide; pNPP, p-nitrophenylphosphate. 0804. E-mail: perlin@phri.nyu.edu. 309 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.