Kinetics of Thapsigargin- Ca-ATPase (Sarcoplasmic Reticulum) Interaction Reveals a Two-step Binding Mechanism and Picomolar Inhibition

doi:10.1074/jbc.270.20.11731

Kinetics of Thapsigargin- Ca-ATPase (Sarcoplasmic Reticulum) Interaction Reveals a Two-step Binding Mechanism and Picomolar Inhibition (*)

From the (1) Medical Research Council Biomembrane Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa

^§To whom correspondence should be addressed.

Abstract

Thapsigargin is a high affinity inhibitor of sarco- and endoplasmic reticulum (SERCA) type ATPases. We have used kinetics to determine the dissociation constant of thapsigargin-sarcoplasmic reticulum Ca-ATPase interaction in the absence and presence of non-ionic detergent. The observed “off” rate constant was measured as 0.0052 s at 26°C by the kinetics of inhibition of ATPase activity following transfer from an inactivated thapsigargin-ATPase complex to native ATPase. Inactive ATPase was produced by cross-linking the active site with glutaraldehyde. The observed dissociation rate constant was increased 7-fold by 0.1% Triton X-100, indicating that perturbation of the transmembrane and stalk region by detergent altered the binding parameters of the inhibitor. In addition, thapsigargin stabilized the ATPase against inactivation caused by detergent in the absence of Ca. The observed “on” rate constant of thapsigargin was measured at 26°C as 25 s irrespective of thapsigargin concentration, by the kinetics of thapsigargin- induced change in intrinsic fluorescence. An Arrhenius plot showed a temperature dependence of this rate constant, indicative of a conformational change in the protein with an activation energy of 9.5 kcal/mol for thapsigargin binding. The affinity of the Ca-ATPase for thapsigargin was calculated to be greater than 2 pM at pH 7.0 and 26°C.

Footnotes

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↵¹ The abbreviations used are:

TG

thapsigargin

SR

sarcoplasmic reticulum

SRV

SR vesicles

MOPS

4-morpholinepropanesulfonic acid

E₁ and E₂

conformations of the non-phosphorylated enzyme in the absence and presence of EGTA, respectively

E₁P and E₂P

phosphorylated forms of E₁ and E₂, respectively

SERCA

sarco- and endoplasmic reticulum.