Potassium-induced changes in phosphorylation and dephosphorylation of (Na+ + K+)-ATPase observed in the transient state.

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.

Section: Kinetics Of Sequential K þ -Mixing Experimentssupporting

confidence: 83%

Section: Kinetics Of Simultaneous K þ -Mixing Experimentsmentioning

confidence: 93%

Section: Kinetics Of Sequential K þ -Mixing Experimentsmentioning

confidence: 99%

See 1 more Smart Citation

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

Myers

Cornelius

Apell

et al. 2011

“…To determine the number of phosphorylation sites per mg of protein each of the enzyme preparations was reacted with 25 M [␥-32 P]ATP at 0°C and pH 7.4 for 20 s in the presence of 150 mM NaCl, 1 mM MgCl 2 , and 30 mM imidazole. Under these K ϩ -free conditions the rate of dephosphorylation is almost two orders of magnitude slower than that of the phosphorylation (Hobbs et al, 1980;Kane et al, 1998), so that ϳ100% of the active Na ϩ ,K ϩ -ATPase molecules become phosphorylated and the amount of radioactive phosphate incorporated closely corresponds to the amount of phosphorylation sites per mg of protein. Phosphorylation was terminated by adding an acid-stop solution at 0°C containing 10% w/v trichloroacetic acid and 2 mM sodium pyrophosphate.…”

Section: N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)-pyridiniummentioning

confidence: 97%

Rate Limitation of the Na+,K+-ATPase Pump Cycle

Lüpfert

Grell

Pintschovius

et al. 2001

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-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 conformation 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 MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation 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 limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.

“…physiological reaction cycle of the Na ϩ ,K ϩ -ATPase. First, in the presence of K ϩ the main reaction route would be via dephosphorylation of E 2 P^E 2 (K 2 ) ϩ P i , which has a forward rate constant of ϳ300 s Ϫ1 (Kane et al, 1998;Mårdh and Zetterqvist, 1974;Hobbs et al, 1980) and the much slower E 2 P^EЈ 2 P transition would be effectively bypassed. Second, in the absence of K ϩ where the dephosphorylation reaction is rate-limiting, no deviations from previous schemes would result because the spontaneous dephosphorylation rate for the two E 2 P phosphoenzymes has been found to be identical (Cornelius et al, 1998) and ϳ1.1 s Ϫ1 at 20°C.…”

Section: Model Simulationsmentioning

confidence: 99%

Rate Determination in Phosphorylation of Shark Rectal Na,K-ATPase by ATP: Temperature Sensitivity and Effects of ADP

Cornelius

1999

Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Na(+) was characterized by chemical quench experiments and by stopped-flow RH421 fluorescence. The appearance of acid-stable phosphoenzyme was faster than the rate of fluorescence increase, suggesting that of the two acid-stable phosphoenzymes formed, RH421 exclusively detects formation of E(2)-P, which follows formation of E(1)-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k(obs) approximately 90 s(-1) and a minor slow phase with a k(obs) of approximately 9 s(-1) (20 degrees C, pH 7.4). The observed rate constants for both the slow and the fast phase could be fitted with identical second-degree functions of the ATP concentration with apparent binding constants of approximately 3.1 x 10(7) M(-1) and 1. 8 x 10(5) M(-1), respectively. Increasing [ADP] decreased k(obs) for the rate of the RH421 fluorescence response to ATP phosphorylation. This could be accounted for by the reaction of ADP with the initially formed E(1)-P followed by a conformational change to E(2)-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K(+) the highly fluorescent E(2)-P is slowly transformed into the "K(+)-insensitive" E'(2)-P subconformation forming a side branch of the main cycle.