Multiple ion-dependent and substrate-dependent sodium/potassium ATPase conformational states. Transient and steady-state kinetic studies

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Abstract
The hydrolysis of .beta.-(2-furyl)acryloyl phosphate (FAP), catalyzed by the Na+/K+-ATPase, is faster than the catalyzed hydrolysis of ATP. This is due to catalyzed hydrolysis of the pseudosubstrate by K+-dependent states of the enzyme, thus bypassing the Na+-dependent enzyme states that are required and are rate limiting in ATP hydrolysis. Unlike ATP, FAP is a positive effector of the E2 state. A study of FAP hydrolysis permits a detailed analysis of later steps in the overall ion translocation-ATP hydrolysis pathway. During the steady state of FAP hydrolysis in the presence of K+, substantial phosphoryl-enzyme is formed, as is indicated by the covalent incorporation of 32P from [32P]FAP. A comparison of the phosphoryl-enzyme yield with the rate of overall hydrolysis reveals that at 25.degree. C the phosphoryl-enzyme formed is all kinetically competent. Both the yield of phosphoryl-enzyme and the rate of overall hydrolysis of FAP are [K+] dependent. The transition E1 .dblarw. E2 is also [K+] dependent, but the rate of transition is differently affected by [K+] than are the above-mentioned two processes. Two distinct roles for K+ are indicated, as an effector of the E1-E2 equilibrium and as a "catalyst" in the hydrolysis of the E2-P. In contrast to the results at 25.degree. C, a virtually stoichiometric yield of phosphoryl-enzyme occurs at 0.degree. C in the presence of Na+ and the absence of K+. At lower concentrations of K+ and in the presence of Na+, the hydrolysis of FAP at 0.degree. C proceeds substantially through the E1-E2 pathway characteristic of ATP hydrolysis. The selectivity of FAP for the E2-K+-dependent pathway is due to the thermal inactivation of E1 at 25.degree. C in the absence of ATP or ATP analogues, even at high concentrations of Na+. These results emphasize the existence of multiple functional "E1" and "E2" states of the overall ATPase-ion translocation pathway.

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