Substrate Regulation of Calcium Binding in Ca2+-ATPase Molecules of the Sarcoplasmic Reticulum

Abstract

The membrane-bound Ca 2ϩ -ATPase (110 kDa) of the sarcoplasmic reticulum (SR) 1 is a calcium pump (1-3) and a P-type ATPase (4, 5). The Ca 2ϩ -ATPase transports 2 mol of calcium across the SR membrane by hydrolytic coupling with 1 mol of ATP, accompanying the transition of the ATPase from E 1 (high affinity state for calcium) to E 2 (low affinity state for calcium) (1-3, 5). The ATP hydrolysis cycle has been shown to be accelerated by ATP binding to a putative regulatory site (1,3,6,7) at concentrations higher than those required for saturation of the catalytic site (8, 9). ATP regulation consists of the acceleration of several intermediate steps in the catalytic reaction (10 -14). Recently, Toyoshima et al. (15) determined the atomic structure of the pump enzyme at a resolution of 2.6 Å and showed that the ATPase molecule has one nucleotide-binding site. However, the molecular basis of the regulatory site has not been clarified (16 -23).Calcium binding to the two calcium transport sites of the enzyme is required for phosphorylation of the enzyme with ATP; the phosphorylation drives the calcium transport reaction (1-3). It is therefore crucial to determine whether calcium binding is regulated by ATP binding. Based on observations of the velocities of calcium transport that are supported by various kinds of high energy phosphate compounds, Ogawa (24) and Ogawa and Ebashi (25) reported that ATP increases the calcium affinity of the enzyme depending on the ATP concentration (0.3 M to 2.7 mM). On the other hand, the calcium dependence of equilibrium calcium binding in the absence of ATP (26) has been shown to exhibit a cooperative profile (Hill number (n H ) of ϳ2) with K 0.5 ϳ 0.4 M. Such a cooperative profile of kinetic calcium binding (n H ϳ 2, K 0.5 ϳ 0.1 M), which was obtained from the calcium dependence of Ca 2ϩ -ATPase activity at a saturating concentration (5 mM) of ATP, has also been observed (27). To clarify whether ATP changes calcium binding of the ATPase, kinetic calcium binding to the enzyme should be examined in the presence and absence of ATP. Recently, the enzyme molecules in the SR membrane have been suggested to exist as two conformational variants of the chemically equivalent enzyme molecules at a ratio of 1:1 (Fig. 1) on the basis of the following observations (28). (i) At 0°C, about half of the calcium-binding sites of the enzyme molecules are in a slow (t1 ⁄2 Ն 2 s)/rapid (t Ͻ2 s) binding state dependent on pH, and the other half are in a slow binding state independent of pH. The enzyme molecules are slowly or rapidly phosphorylated by ATP, concurrent with slow or rapid calcium binding at a ratio of ϳ1:2, indicating that each of the two pools of the calcium sites belongs to one of the two different types (A and B forms) of the enzyme molecules, which are in pH-dependent equilibrium between E 1 and E 2 and predominantly in E 2 independent of pH, respectively, before calcium binding (Fig. 2). (ii) The amino acid sequence of lysyl endopeptidase peptides of the enzyme preparation is homogeneous ...

Replacement of Several Single Amino Acid Side Chains Exposed to the Inside of the ATP-binding Pocket Induces Different Extents of Affinity Change in the High and Low Affinity ATP-binding Sites of Rat Na/K-ATPase

“…One accepts ATP with a high affinity to form EP and the other, in the adjacent ␣-chain, accepts ATP with a low affinity to accelerate Na ϩ /K ϩ -ATPase activity. The present finding is consistent with the view that Na ϩ /K ϩ -ATPase functions out of phase (5-7, 21, 23, 47, 48) as a diprotomer, (␣␤) 2 , or a much higher oligomer, (␣␤) 4 , as in the case of cross-talking gastric H ϩ /K ϩ -ATPase (45) and possibly Ca 2ϩ / H ϩ -ATPase (49,50).…”

Substrate Regulation of Calcium Binding in Ca2+-ATPase Molecules of the Sarcoplasmic Reticulum

“…1 in the accompanying article (32)). This model was hypothesized on the basis of the following observations (18). (i) There are two types of enzyme molecules that are in pH-dependent equilibrium between E 1 and E 2 and predominantly in E 2 independent of pH, respectively, before calcium binding.…”

“…In the first part of this study on substrate regulation of calcium binding (32), the regulatory effect of ATP on calcium binding was examined, and ATP binding to the regulatory site of the enzyme was found to raise the calcium binding performance of the enzyme molecules depending on the ATP concentration, pH, and aggregation state of the molecules. Here, we examined the regulatory effects of CTP, GTP, ITP, and UTP on calcium binding of the enzyme molecules by comparing the calcium dependence of the Ca 2ϩ -activated hydrolysis activity (Ca 2ϩ -NTPase activity) of the NTPs of the enzyme with that of calcium binding in the absence of these NTPs.…”

“…Here, we examined the regulatory effects of CTP, GTP, ITP, and UTP on calcium binding of the enzyme molecules by comparing the calcium dependence of the Ca 2ϩ -activated hydrolysis activity (Ca 2ϩ -NTPase activity) of the NTPs of the enzyme with that of calcium binding in the absence of these NTPs. The results are discussed based on a model of two conformational variants (A and B forms) of chemically equivalent ATPase molecules (18), as was done in the first part of this study (32); the two forms exist in the SR membrane at a ratio of 1:1 and bind two calcium ions in a different manner (see Fig. 1 in the accompanying article (32)).…”

“…The results are discussed based on a model of two conformational variants (A and B forms) of chemically equivalent ATPase molecules (18), as was done in the first part of this study (32); the two forms exist in the SR membrane at a ratio of 1:1 and bind two calcium ions in a different manner (see Fig. 1 in the accompanying article (32)). This model was hypothesized on the basis of the following observations (18).…”