title: Allosteric regulation of proton translocation by a vacuolar adenosinetriphosphatase.
abstract: The kinetics of nucleoside-triphosphate-dependent proton translocation by a vacuolar-type adenosine-triphosphatase have been studied, using the enzyme from bovine chromaffin-granule membranes, purified and reconstituted into proteoliposomes. The reaction was followed by recording the quenching of the fluorescence of the permeant weak base 9-amino-6-chloro-2-methoxyacridine; fluorescence data were collected and stored in digital form, and the initial reaction rates estimated by linear regression. In the absence of nucleoside diphosphate, the dependence of initial rates of proton translocation on substrate concentration were fitted well by the Michaelis-Menten equation, as were the kinetics of ATP hydrolysis. ADP and other nucleoside diphosphates were potent inhibitors of the ATPase, effecting a reduction in the maximum velocity of the reaction, and producing sigmoid substrate-saturation curves which could be fitted by the empirical Hill equation, the Hill coefficient approaching 2 at high inhibitor concentrations. Data sets containing initial-rate estimates were collected over a wide range of independently varied concentrations of substrate and inhibitor and were modeled, using rate equations derived from several different models based on the concerted-transition model of allosteric inhibition proposed by Monod, Wyman and Changeux. These equations were fitted to the data by weighted non-linear regression, using an iterative computer program to obtain the best estimates of kinetic parameters. One model consistently fitted all of the data sets better than all the others, and this model was based on the following assumptions: that the ATPase exists in two conformational states, R and T; that only the R state is catalytically active; that each state contains three kinetically equivalent catalytic sites, and one regulatory site; that nucleoside triphosphates bind only to the catalytic sites, and that nucleoside diphosphates bind both to the catalytic sites and to the regulatory site. The optimized values of the kinetic parameters indicate that in the absence of nucleoside diphosphate, the enzyme is almost completely in the R state; that nucleoside triphosphates bind more tightly to the R than to the T state; that binding of nucleoside diphosphates to the regulatory site is very tight, but occurs only in the T state; and that competitive binding of nucleoside diphosphates at the catalytic sites is stronger in the T state than in the R state. Experiments conducted with varying total magnesium concentrations indicated that the magnesium complexes of nucleoside diphosphates are much stronger inhibitors than the free nucleotides, and that free nucleoside triphosphates are weakly inhibitory, probably competing with the magnesium complexes for binding at the catalytic sites.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biochemistry, University of Edinburgh, Scotland.
authors.forename: L C
authors.initials: LC
authors.lastname: Webster
authors.forename: J R
authors.initials: JR
authors.lastname: Pérez-Castiñeira
authors.forename: G L
authors.initials: GL
authors.lastname: Atkins
authors.forename: D K
authors.initials: DK
authors.lastname: Apps
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000609:Aminoacridines; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D009711:Nucleotides / Q000494:pharmacology; D011510:Proteolipids; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D013050:Spectrometry, Fluorescence; D014617:Vacuoles / Q000378:metabolism
chemical_list: D000609:Aminoacridines; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009711:Nucleotides; D011510:Proteolipids; D011522:Protons; C020485:proteoliposomes; C010847:9-amino-6-chloro-2-methoxyacridine; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases