肌浆网钙离子转运三磷酸腺苷酶的锂-7核磁共振、水质子核磁共振和钆电子顺磁共振研究

Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance, and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase.

作者信息

Stephens E M, Grisham C M

出版信息

Biochemistry. 1979 Oct 30;18(22):4876-85. doi: 10.1021/bi00589a016.

Abstract

The interactions of gadolinium ion, lithium, and two substrate analogues, beta,gamma-imido-ATP (AMP-PNP) and tridentate CrATP, with the calcium ion transport adenosine triphosphatase (Ca2+-ATPase) of rabbit muscle sarcoplasmic reticulum have been examined by using 7Li+ NMR, water proton NMR, and Gd3+ EPR studies. Steady-state phosphorylation studies indicate that Gd3+ binds to the Ca2+ activator sites on the enzyme with an affinity which is approximately 10 times greater than that of Ca2+. 7Li+, which activates the Ca2+-ATPase in place of K+, has been found to be a suitable nucleus for probing the active sites of monovalent cation-requiring enzymes. 7Li+ nuclear relaxation studies demonstrate that the binding of Gd3+ ion to the two Ca2+ sites on Ca2+-ATPase increases the longitudinal relaxation rate (1/T1) of enzyme-bound Li+. The increase in 1/T1 was not observed in the absence of enzyme, indicating that the ATPase enhances the parmagnetic effect of Gd3+ on 1/T1 of 7Li+. Water proton relaxation studies also show that the ATPase binds Gd3+ at two tight-binding sites. Titrations of Gd3+ solutions with Ca2+-ATPase indicate that the tighter of the two Gd3+-binding sites (site 1) provides a ghigher enhancement of water relaxation than the other, weaker Gd3+ site (site 2) and also indicate that the average of the enhancements at the two sites is 7.4. These data, together with a titration of the ATPase with Gd3+ ion, yield enhancements, epsilonB, of 9.4 at site 1 and 5.4 at site 2. Analysis of the frequency dependence of 1/T1 of water indicates that the electron spin relaxation taus of Gd3+ is unusually long (2 X 10(-9) s) and suggests that the Ca2+-binding sites on the ATPase experience a reduced accessiblity of solvent water. This may indicate that the Ca2+ sites on the Ca2+-ATPase are buried or occluded within a cleft or channel in the enzyme. The analysis of the frequency dependence is also consistent with three exchangeable water protons on Gd3+ at site 1 and two fast exchanging water protons at site 2. Addition of the nonhydrolyzing substrate analogues, AMP-PNP and tridenate CrATP, to the enzyme-Gd3+ complex results in a decrease in the observed enhancement, with little change in the dipolar correlation time for Gd3+, consistent with a substrate-induced decrease in the number of fast-exchanging water protons on enzyme-bound Gd3+. From the effect of Gd3+ on 1/T1 of enzyme-bound Li+, Gd3+-Li+ separations of 7.0 and 9.1 A are calculated. On the assumption of a single Li+ site on the enzyme, these distances set an upper limit on the separation between Ca2+ sites on the enzyme of 16.1 A.

摘要

通过使用(^{7}Li^{+})核磁共振（NMR）、水质子NMR和钆（(Gd^{3+})）电子顺磁共振（EPR）研究，考察了钆离子、锂以及两种底物类似物β,γ-亚氨基三磷酸腺苷（AMP-PNP）和三齿铬三磷酸腺苷（CrATP）与兔肌肉肌浆网钙离子转运三磷酸腺苷酶（(Ca^{2+})-ATPase）的相互作用。稳态磷酸化研究表明，(Gd^{3+})以大约比(Ca^{2+})高10倍的亲和力结合到酶上的(Ca^{2+})激活位点。已发现(^{7}Li^{+})能替代(K^{+})激活(Ca^{2+})-ATPase，是探测需要单价阳离子的酶活性位点的合适原子核。(^{7}Li^{+})核弛豫研究表明，(Gd^{3+})离子与(Ca^{2+})-ATPase上的两个(Ca^{2+})位点结合会增加酶结合的(Li^{+})的纵向弛豫率（(1/T_{1})）。在没有酶的情况下未观察到(1/T_{1})的增加，这表明ATPase增强了(Gd^{3+})对(^{7}Li^{+})的(1/T_{1})的顺磁效应。水质子弛豫研究还表明，ATPase在两个紧密结合位点结合(Gd^{3+})。用(Ca^{2+})-ATPase滴定(Gd^{3+})溶液表明，两个(Gd^{3+})结合位点中较紧密的位点（位点1）比另一个较弱的(Gd^{3+})位点（位点2）对水弛豫的增强作用更大，并且还表明两个位点增强作用的平均值为7.4。这些数据，连同用(Gd^{3+})离子滴定ATPase的数据，得出位点1的增强因子(\epsilon_{B})为9.4，位点2为5.4。对水质子(1/T_{1})的频率依赖性分析表明，(Gd^{3+})的电子自旋弛豫时间(\tau_{s})异常长（(2×10^{-9} s)），这表明ATPase上的(Ca^{2+})结合位点周围溶剂水的可及性降低。这可能表明(Ca^{2+})-ATPase上的(Ca^{2+})位点被埋在酶中的裂缝或通道内或被其封闭。频率依赖性分析还与位点1上(Gd^{3+})的三个可交换水质子和位点2上的两个快速交换水质子一致。向酶-(Gd^{3+})复合物中加入非水解底物类似物AMP-PNP和三齿CrATP会导致观察到的增强作用降低，而(Gd^{3+})的偶极相关时间变化很小，这与底物诱导的酶结合的(Gd^{3+})上快速交换水质子数量减少一致。根据(Gd^{3+})对酶结合的(Li^{+})的(1/T_{1})的影响，计算出(Gd^{3+})与(Li^{+})的距离分别为7.0和9.1 Å。假设酶上有一个单一的(Li^{+})位点，这些距离确定了酶上(Ca^{2+})位点之间的最大距离为16.1 Å。