Kinetics of K+ Occlusion by the Phosphoenzyme of the Na+,K+-ATPase

Abstract

Investigations of K+-occlusion by the phosphoenzyme of Na+,K+-ATPase from shark rectal gland and pig kidney by stopped-flow fluorimetry reveal major differences in the kinetics of the two enzymes. In the case of the pig enzyme, a single K+-occlusion step could be resolved with a rate constant of 342 (±26) s−1. However, in the case of the shark enzyme, two consecutive K+-occlusions were detected with rate constants of 391 (±19) s−1 and 48 (±2) s−1 at 24°C and pH 7.4. A conformational change of the phosphoenzyme associated with K+-occlusion is, thus, the major rate-determining step of the shark enzyme under saturating concentrations of all substrates, whereas for the pig enzyme the major rate-determining step under the same conditions is the E2 → E1 transition and its associated K+ deocclusion and release to the cytoplasm. The differences in rate constants of the K+ occlusion reactions of the two enzymes are paralleled by compensating changes to the rate constant for the E2 → E1 transition, which explains why the differences in the enzymes' kinetic behaviors have not previously been identified.