Transient and steady-state kinetic studies of sodium-potassium adenosine triphosphatase using beta-(2-furyl)acryloyl phosphate as chromophoric substrate assay.
A convenient and highly specific continuous spectrophotometric assay for sodium-potassium adenosine triphosphate 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. These results indicate that the pathway demonstrated with ATP is not appropriate to the FAPase mechanism. The results suggest that acyl phosphates are 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.
D000179:Acrylates; D000818:Animals; D004524:Eels; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D009943:Organophosphorus Compounds / Q000138:chemical synthesis; D011188:Potassium / Q000494:pharmacology; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D000179:Acrylates; D009943:Organophosphorus Compounds; C025983:beta-(2-furyl)acryloyl phosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium