Temperature dependence of the rates of conformational changes reported by fluorescein 5'-isothiocyanate modification of H+,K(+)- and Na+,K(+)-ATPases.
Stopped-flow fluorometry has been used to measure the forward and reverse rates of the conformational change from E1 to E2 in the fluorescein-modified proton and sodium pumps (1) as a function of Na+ and K+ concentrations to verify the proposed mechanism of ion interaction with the enzymes and (2) as a function of temperature to gain insight into the nature of the conformational transition. (1) The fluorescence changes caused by Na+ and K+ are consistent with rapid competitive binding of the two ions to the E1 conformations of the enzymes followed by rate-limiting transitions between E1K and E2K. (2) Reaction coordinate diagrams for the E1K to E2K transitions in the H,K-ATPase and Na,K-ATPase are qualitatively similar. Enthalpy barriers to reaction are partially compensated by increased entropy in the transition states. However, there are striking quantitative differences between the two enzymes. The E2K to E1K reaction of the H,K-ATPase is more than 2 orders of magnitude faster (tau 1/2 = 6 ms at 22 degrees C) than the reverse rate of the Na,K-ATPase transition (tau 1/2 = 1.6 s), explaining repeated failure to detect a K(+)-"occluded" form of the H,K-enzyme. The E2K conformer of the Na,K-ATPase is 3 orders of magnitude more stable than E1K, while the E1K and E2K conformations of the H,K-ATPase are nearly equivalent energetically.
Faller
L D
LD
Department of Medicine, University of California, Los Angeles School of Medicine.
Diaz
R A
RA
Scheiner-Bobis
G
G
Farley
R A
RA
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D012964:Sodium / Q000494:pharmacology; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013552:Swine; D013816:Thermodynamics; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D015222:Sodium Channels; D013861:Thiocyanates; D012964:Sodium; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D011188:Potassium