Andersen J P, Sorensen T L, Povlsen K, Vilsen B
Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark.
J Biol Chem. 2001 Jun 29;276(26):23312-21. doi: 10.1074/jbc.M102384200. Epub 2001 Apr 23.
The specific functional roles of various parts of the third transmembrane segment (M3) of the sarcoplasmic reticulum Ca(2+)-ATPase were examined by functionally characterizing a series of mutants with multiple or single substitutions of M3 residues. Steady-state and transient kinetic measurements, assisted by computer simulation of the time and Ca(2+) dependences of the phosphorylation level, were used to study the partial reaction steps of the enzyme cycle, including the binding and dissociation of Ca(2+) at the high affinity cytoplasmically facing sites. The mutation Lys-Leu-Asp-Glu(255) --> Glu-Ile-Glu-His resulted in a conspicuous increase in the rate of Ca(2+) dissociation as well as a displacement of the major conformational equilibria of the phosphoenzyme and dephosphoenzyme forms. The point mutant Phe(256) --> Ala also showed an increased rate of Ca(2+) dissociation, whereas a conspicuous decrease both in the rate of Ca(2+) dissociation and in the rate of Ca(2+) binding was found for the mutant Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile. These findings suggest that the NH(2)-terminal half of M3 is involved in control of the gateway to the Ca(2+) sites. The main effect of two mutations to the COOH-terminal half of M3, Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val and Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe-Ile-Leu, was a block of the dephosphorylation.
通过对肌浆网Ca(2+)-ATP酶第三跨膜片段（M3）各部分进行一系列多个或单个残基取代的突变体进行功能表征，研究了M3不同部分的具体功能作用。借助对磷酸化水平的时间和Ca(2+)依赖性进行计算机模拟，采用稳态和瞬态动力学测量方法，研究了酶循环的部分反应步骤，包括Ca(2+)在面向细胞质的高亲和力位点的结合和解离。Lys-Leu-Asp-Glu(255) --> Glu-Ile-Glu-His突变导致Ca(2+)解离速率显著增加，同时磷酸化酶和脱磷酸化酶形式的主要构象平衡发生位移。点突变Phe(256) --> Ala也显示出Ca(2+)解离速率增加，而突变Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile的Ca(2+)解离速率和Ca(2+)结合速率均显著降低。这些发现表明，M3的NH(2)-末端一半参与了对Ca(2+)位点通道的控制。对M3的COOH-末端一半进行的两个突变Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val和Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe-Ile-Leu的主要作用是阻断脱磷酸化。
