MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis.
Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K(m) values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.
Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA. milgromy@upstate.edu
Elena M
EM
Milgrom
Yakov M
YM
Milgrom
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000737:chemistry; D006868:Hydrolysis; D009578:Nitrobenzenes / Q000737:chemistry*; D010080:Oxazoles / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry*
C511330:7-chloro-4-nitrobenz-2-oxa-1,3-diazole; D004791:Enzyme Inhibitors; D009578:Nitrobenzenes; D010080:Oxazoles; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan