INHIBITION OF PHOSPHOENZYME FORMATION FROM PHOSPHATE AND SARCOPLASMIC-RETICULUM CA2+-ATPASE BY VANADATE BINDING TO HIGH-AFFINITY OR LOW-AFFINITY SITE ON THE ENZYME

In the preceding paper, we suggested that 1 mol Ca2+-ATPase of sarcoplasmic reticulum (SR) contains 0.5 mol of high-affinity vanadate binding sites as well as 0.5 mol of low-affinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (P(i)). When vanadate was added to the reaction medium in which the Ca2+-ATPase had been phosphorylated by P(i) in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17 muM, and reached maximum at 50 muM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of E2P was competitively prevented by P(i) with a dissociation constant of 7.4 mM, which was very close to K(m) for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with P(i) at a phosphorylation site on the Ca2+-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca2+-ATPase. When SR was incubated with 5 muM vanadate prior to the phosphorylation, the amount of E2P decreased to below 5% of the initial level in 10 min. Since vanadate binding to the low-affinity site was negligibly small at this concentration, the inhibition of E2P formation was considered to be caused by vanadate binding to the high-affinity site. P(i) also interrupted the slow decay of E2P which was induced by vanadate binding to the high-affinity site. The apparent dissociation constant of P(i) binding under this condition was estimated to be about 0.4 mM which is much lower than the K(m) value for the E2P formation. Based on these results, we discuss the involvement of dimeric interaction of the Ca2+-ATPase in the E2P formation.