Transient and steady-state kinetic studies of sodium-potassium adenosine triphosphatase using .beta.-(2-Furyl)acryloyl phosphate as chromophoric substrate assay

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Abstract
A convenient and highly specific continuous spectrophotometric assay for (Na,K)-ATPase activity utilizing the rapidly hydrolyzed and high-affinity chromophoric substrate .beta.-(2-furyl)acryloyl phosphate (FAP) is described. The (Na, K)-ATPase-catalyzed hydrolysis of FAP is faster than that for ATP under all ionic conditions. The rate is neither inhibited nor activated by Na+; it is dependent on [K+] and on [Mg2+]. The hydrolysis of FAP to furylacrylate is accompanied by a large shift in the UV absorbance maximum. The spectrum of FAP, but not furylacrylate, is sensitive to noncovalent ligation with Mg2+, a happenstance which permits the identification of Mg2+FAP, and consequently allows for a probe of the role of Mg2+ in the catalysis. Mg2+ binding to the active site is essential for catalysis. MgFAP is more tightly bound to the site than is FAP2-, but the complex is not obligatory for catalysis. The formation of a phosphoryl-enzyme intermediate is not evident in the reaction of FAP with the enzyme. Transient kinetic experiments, utilizing an excess of MgFAP, demonstrate a unique steady-state rate-limiting production of furylacrylate. The pathway demonstrated with ATP is probably not appropriate to the FAPase mechanism. Acyl phosphates may be good phosphatase substrates either because they are analogues of the phosphatase-specific phosphoryl-enzyme or because they react exclusively with the isomerized E2 form of the enzyme. [Enzyme preparations from sheep kidney and the electric organ of Torpedo californica and Electrophorus electricus were used.].

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