Evidence Calcium Pump Binds Magnesium before Inorganic Phosphate

Abstract

Calcium pump-catalyzed (18)O exchange between inorganic phosphate and water was studied to test the hypothesis that all P-type pumps bind Mg(2+) before P(i) and validate utilization of the rate equation for ordered binding to interpret differences between site-directed mutants and wild-type enzyme. The results were remarkably similar to those obtained earlier with sodium pump (Kasho, V. N., Stengelin, M., Smirnova, I. N., and Faller, L. D. (1997) Biochemistry 36, 8045- 8052). The equation for ordered binding of Mg(2+) before P(i) fit the data best with only a slight chance (0.6%) of P(i) binding to apoenzyme. Therefore, P(i) is the substrate, and Mg(2+) is an obligatory cofactor. The intrinsic Mg(2+) dissociation constant from metalloenzyme (K(M) = 3.5 +/- 0.3 mm) was experimentally indistinguishable from the sodium pump value. However, the half-maximal concentration for P(i) binding to metalloenzyme ((K(p)(')=6.3+/-0.6 mM)) was significantly higher ( approximately 6-fold), and the probability of calcium pump forming phosphoenzyme from bound P(i) (P(c) = 0.04 +/- 0.03) was significantly lower ( approximately 6-fold) than for the sodium pump. From estimates of the rate constants for phosphorylation and dephosphorylation, the calcium pump appears to catalyze phosphoryl group transfer less efficiently than the sodium pump. Ordered binding of Mg(2+) before P(i) implies that both calcium pump and sodium pump form a ternary enzyme.metal.phosphate complex, consistent with molecular structures of other haloacid dehalogenase superfamily members that were crystallized with Mg(2+) and phosphate, or a phosphate analogue, bound.