Rate limitation of the Na+,K+-ATPase pump cycle

The kinetics of Na-dependent phosphorylation of the Na+,K+-ATPase by ATP we re investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na+], and [Mg2+], pH 7.4, and 24 degreesC). The w ell-established effect of buffer composition on the E-2-E-1 equilibrium was used as a tool to investigate the effect of the initial enzyme conformatio n on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E- 2) yielded a 1/tau value of 59 s(-1). Addition of MgCl2 (5 mM), NaCl (2 mM) , or ATP (2 mM) to the preincubation solution resulted in increases in 1/ta u to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosp horylation from the E-2 state to an E-1 or E-1-like state. The results thus demonstrate conclusively that the E-2 --> E-1 transition does in fact limi t the rate of subsequent reactions of the pump cycle. Based on the experime ntal results, the rate constant of the E-2 --> E-1 transition under physiol ogical conditions could be estimated to be similar to 65 s(-1) for pig kidn ey enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account t he rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (similar to 48 s(-1) and similar to 43 s(-1) for pig and rabbit, respectively) calculat ed from steady-state measurements. For enzyme of the a, isoform the E-2 --> E-1 conformational change is thus shown to be the major rate-determining s tep of the entire enzyme cycle.