Identification of potential regulatory sites of the Na+,K+-ATPase by kinetic analysis.
Kinetic models are presented that allow the Na(+),K(+)-ATPase steady-state turnover number to be estimated at given intra- and extracellular concentrations of Na(+), K(+), and ATP. Based on experimental transient kinetic data, the models utilize either three or four steps of the Albers-Post scheme, that is, E(2) --> E(1), E(1) --> E(2)P (or E(1) --> E(1)P and E(1)P --> E(2)P), and E(2)P --> E(2), which are the major rate-determining steps of the enzyme cycle. On the time scale of these reactions, the faster binding steps of Na(+), K(+), and ATP to the enzyme are considered to be in equilibrium. Each model was tested by comparing calculations of the steady-state turnover from rate constants and equilibrium constants for the individual partial reactions with published experimental data of the steady-state activity at varying Na(+) and K(+) concentrations. To provide reasonable agreement between the calculations and the experimental data, it was found that Na(+)/K(+) competition for cytoplasmic binding sites was an essential feature required in the model. The activity was also very dependent on the degree of K(+)-induced stimulation of the reverse reaction E(1) --> E(2). Taking into account the physiological substrate concentrations, the models allow the most likely potential sites of short-term Na(+),K(+)-ATPase regulation to be identified. These were found to be (a) the cytoplasmic Na(+) and K(+) binding sites, via changes in Na(+) or K(+) concentration or their dissociation constants, (b) ATP phosphorylation (as a substrate), via a change in its rate constant, and (c) the position of the E(2)<==>E(1) equilibrium.
School of Chemistry, University of Sydney, Sydney, New South Wales 2006, Australia.
Benjamin Y
BY
Kong
Ronald J
RJ
Clarke
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002414:Cations, Monovalent / Q000737:chemistry; D002417:Cattle; D003198:Computer Simulation; D004789:Enzyme Activation; D005110:Extracellular Space / Q000201:enzymology; D007424:Intracellular Fluid / Q000201:enzymology; D007700:Kinetics; D008297:Male; D008956:Models, Chemical; D010766:Phosphorylation; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
D002414:Cations, Monovalent; D021122:Protein Subunits; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium