钙离子从肌浆网磷酸化钙转运三磷酸腺苷酶上的解离：与磷酸化酶结合的钙离子的动力学等效性

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme.

作者信息

Hanel A M, Jencks W P

机构信息

Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.

出版信息

Biochemistry. 1991 Nov 26;30(47):11320-30. doi: 10.1021/bi00111a019.

DOI:10.1021/bi00111a019

PMID:1835656

Abstract

The internalization of 45Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25 degrees C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS.Tris, pH 7.0). Intact vesicles containing either 10-20 microM or 20 mM Ca2+ were preincubated with 45Ca for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of Etot with the rate constant k = 300 s-1. The two 45Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and 45Ca with excess 40Ca, or by mixing the enzyme and 40Ca with 45Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with kc = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater than or equal to 10(5) s-1) than the forward and reverse reactions for the conformational change (k-c = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (kmix greater than or equal to 10(5) s-1) before dissociating. A Hill slope of nH = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.

摘要

在酶的单次周转过程中，通过加入7 mM ADP和EGTA淬灭反应（25℃，5 mM MgCl₂，100 mM KCl，40 mM MOPS - Tris，pH 7.0），测定了兔肌肉钙转运ATP酶将⁴⁵Ca内化到肌浆网囊泡中的情况。含有10 - 20 μM或20 mM Ca²⁺的完整囊泡先与⁴⁵Ca预孵育约20秒，然后与0.20 - 0.25 mM ATP和过量EGTA混合，以使总酶（Etot）的磷酸化程度达到70%，速率常数k = 300 s⁻¹。与磷酸酶（EP）结合的两个⁴⁵Ca离子在被内化时，随着一级反应（速率常数k = 约30 s⁻¹）进行，对ADP淬灭变得不敏感。在加入ATP和EGTA之前，通过将酶与⁴⁵Ca和过量⁴⁰Ca混合，或通过将酶与⁴⁰Ca和⁴⁵Ca混合50毫秒，选择性地标记了与游离酶结合的第一个和第二个Ca²⁺离子。每个离子向负载或空囊泡中的内化遵循一级动力学，k = 约30 s⁻¹；没有迹象表明两种Ca²⁺离子内化存在双相动力学或诱导期。囊泡内20 mM Ca²⁺的存在对单个离子或两个离子的内化动力学或程度均无影响。与磷酸酶结合的Ca²⁺离子在动力学上是等效的。单个Ca²⁺离子内化的一级反应与磷酸酶的限速构象变化一致，kc = 30 s⁻¹，随后Ca²⁺离子从E≈P·Ca₂上独立的不同结合位点快速解离；腔内钙不抑制钙从这些位点的解离。或者，Ca²⁺离子可能在限速构象变化后从E≈P·Ca₂依次解离。然而，单个离子的解离顺序无法区分。有序顺序解离机制要求离子解离速度比构象变化的正向和逆向反应速度快得多（k≥10⁵ s⁻¹）（k - c = 约3000 s⁻¹）。最后，Ca²⁺离子在解离前可能在磷酸酶上快速交换位置（kmix≥10⁵ s⁻¹）。通过在六种不同Mg²⁺浓度下进行速率测量，得到Ca²⁺与磷酸酶低亲和力转运位点结合对周转抑制的Hill斜率nH = 1.0 - 1.2，K0.5 = 0.8 - 0.9 mM。