Interaction of a K(+)-competitive inhibitor, a substituted imidazo[1,2a] pyridine, with the phospho- and dephosphoenzyme forms of H+, K(+)-ATPase.

Abstract

Defining the structural and catalytic properties of the ion transport site(s) of enzyme-phosphorylating ATPases is of key importance in understanding the mechanism of ion transport by these enzymes. In the case of the H+, K(+)-ATPase, SCH 28080 (3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a]-pyridine) has been shown to act as a high affinity, extracytosolic, K(+)-competitive inhibitor of Mg2+, K(+)-ATPase activity (Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson, R., Mendlein, J., Rabon, E., and Sachs, G. (1987) J. Biol. Chem. 262, 2077-2084). To define the nature of the SCH 28080-binding site in relation to the catalytic cycle of the enzyme, we have investigated the effects of this potential K+ transport site probe on the steady-state and partial reactions of the H+, K(+)-ATPase. In the absence of K+, SCH 28080 inhibits Mg2(+)-ATPase activity with high affinity (apparent Ki = 30 nM). Inhibition is due to K(+)-like prevention of phosphoenzyme formation. SCH 28080 has no effect on Mg2(+)-catalyzed dephosphorylation. SCH 28080, at concentrations less than 0.5 microM, increases the apparent Km for K+ for Mg2+, K(+)-ATPase activity with little effect on the maximum velocity. At higher concentrations of SCH 28080, reversal of inhibition by higher K+ concentrations is not complete, due to inhibition of ATPase activity by high K+. In contrast, SCH 28080 inhibits K(+)-stimulated dephosphorylation by competitively displacing K+ from phosphoenzyme with an extracytosolic conformation of the monovalent cation site (E2P) at low concentrations of SCH 28080 and K+. At higher concentrations, 10 microM SCH 28080 and 50 mM K+, a slowly dephosphorylating complex with both SCH 28080 and K+ bound to E2P may form which represents a small fraction of the total E2P (15-25%). Preincubation of SCH 28080 with E2P completely blocks K(+)-stimulated dephosphorylation, and K+ is unable to reverse this preincubation effect, indicating that the SCH 28080 dissociation rate is at least as slow as K(+)-independent dephosphorylation of E2P. These findings indicate that SCH 28080 inhibits K(+)-stimulated ATPase activity by competing with K+ for binding to E2P and blocking K(+)-stimulated dephosphorylation. In the absence of K+, SCH 28080 has a higher apparent affinity for E2P, but it permits K(+)-independent dephosphorylation. Since the dissociation rate of SCH 28080 from the enzyme is slow, phosphoenzyme formation is prevented by SCH 28080 remaining bound to the extracytosolic conformation of the monovalent cation site, thereby reducing the steady-state level of phosphoenzyme.