Role of Mg2+ ions in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na+,K(+)-ATPase.
The role of Mg2+ in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na,K-ATPase has been studied by stopped-flow fluorometry. K+ causes a fluorescence quench that is reversed by Na+. The principal experimental observations are as follows: (1) Mg2+ decreases the apparent affinity of the enzyme for K+ but does not affect the maximum rate of the K+ quench. (2) The amplitude of the K+ quench depends hyperbolically on the K+ concentration, and the maximum amplitude is unaffected by the Mg2+ concentration. (3) The rate at which Na+ reverses the K+ quench depends inversely on the Mg2+ concentration. (4) The amplitude of the Na+ reversal also decreases with increasing Mg2+ concentration. The data are quantitatively explained by a model that assumes only two enzyme conformations, detectable by their fluorescence emission. Mg2+ increases Kd for K+ from 14 to 223 mM. At 22 degrees C, Kd = 0.16 mM for Mg2+ dissociation from E1, and the heat of Mg2+ binding, delta H degrees, is 11.4 kcal mol-1. Kd is more than an order of magnitude larger for Mg2+ dissociation from E2K. Mg2+ binding does not affect the forward (E1K-->E2K) rate constant (kf), but decreases the reverse rate constant (kr) thus increasing the equilibrium constant for the reaction (Kc = kf/kr) 6-fold. Therefore, Mg2+ is not directly involved in the conformational transition, but the study supports proposals that Mg2+ binding and release may help to regulate the transport cycle by shifting the distribution of enzyme between E1 and E2 conformers.
Department of Medicine, University of California, Los Angeles School of Medicine.
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Smirnova
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Faller
D000818:Animals; D016650:Fluorescein-5-isothiocyanate; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000502:physiology*; D008956:Models, Chemical; D011188:Potassium; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D011188:Potassium