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.

Identification of Electric-Field-Dependent Steps in the Na+,K+-Pump Cycle

“…This equation is based on a model in which a fluorescence change is assumed to occur only after two K þ ions have already bound to the E2P state of the enzyme. This is consistent with recent stopped-flow results of Myers et al (14), in which only a single kinetic phase of K þ occlusion was observed although it is known that two K þ ions are occluded. An attempt was also made to fit two identical independent binding-site models to the experimental data, but the goodness-of-fit was much improved by use of Eq.…”

“…The release of Na þ ions from the phosphorylated form of the protein causes a significant increase in the fluorescence of RH421 (24,26,29). The interaction of K þ ions with the phosphorylated form of the protein causes a significant drop in the fluorescence of RH421 (14,30,31). Therefore, if binding alone of Na þ or K þ to ion transport sites on the E2P state of the protein (i.e., before occlusion) were causing a change in intramembrane electric-field strength detectable by RH421, one would expect also binding of BTEA to the protein to cause a drop in fluorescence, not an increase.…”

“…Styrylpyridinium dyes of this type were first used to follow the kinetics of partial reactions of the Na þ ,K þ -ATPase by Klodos and Forbush (22,23), and have been widely used since that time (13,(24)(25)(26)(27)(28)(29). If ion binding is a voltagedependent reaction and causes a change in local electric-field strength in the adjacent membrane, one would expect binding of BTEA to the Na þ ,K þ -ATPase to cause a drop in RH421 fluorescence, as has been observed after addition of K þ (14,30,31). In fact, as we will show here, this is not what is observed; only occludable ions (K þ , Rb þ , or Cs þ ) cause a drop in fluorescence.…”

“…These results suggest that extracellular Na þ deocclusion is associated with movement of charge. More recently, Myers et al (14) found that the membrane-bound voltage-sensitive fluorescent probe RH421 (closely related to di-8-ANEPPS) responds to K þ occlusion by the E2P form of the enzyme with a fluorescence change, thus suggesting that K þ occlusion from the extracellular medium involves charge movement. Thus, measurements on the native enzyme in intact cells and purified enzyme in open membrane fragments seem to be yielding different information concerning the reaction steps in which charge movement is occurring.…”

Estudo bioquímico comparativo da (Na+ , K+)-ATPase branquial de Macrobrachium amazonicum (Heller, 1862) habitante de regiões continentais

“…A desfosforilação (etapa ) leva a enzima de volta à forma E2(2K) (Myers et al, 2011), reiniciando o ciclo catalítico.…”