Rate limitation of the Na(+),K(+)-ATPase pump cycle.
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.
Lüpfert
C
C
School of Chemistry, University of Sydney, Sydney NSW 2006, Australia.
Grell
E
E
Pintschovius
V
V
Apell
H J
HJ
Cornelius
F
F
Clarke
R J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D003198:Computer Simulation; D004492:Edetic Acid / Q000378:metabolism* / Q000494:pharmacology; D005456:Fluorescent Dyes; D006639:Histidine / Q000378:metabolism* / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism* / Q000494:pharmacology; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D011817:Rabbits; D012965:Sodium Chloride / Q000378:metabolism* / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D002021:Buffers; D005456:Fluorescent Dyes; D015636:Magnesium Chloride; D012965:Sodium Chloride; D006639:Histidine; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D000254:Sodium-Potassium-Exchanging ATPase