Vilsen B, Andersen J P, Clarke D M, MacLennan D H
Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, Ontario, Canada.
J Biol Chem. 1989 Dec 15;264(35):21024-30.
Site-specific mutagenesis was used to investigate whether Pro160, Pro195, Pro308, Pro312, Pro803, and Pro812 play essential roles in the function of the sarcoplasmic reticulum Ca2(+)-ATPase. All six prolines were substituted with alanine; and in addition, Pro308 was replaced by glycine and Pro312 by glycine as well as by leucine. Mutant cDNAs were expressed in COS-1 cells, and mutant Ca2(+)-ATPases located in the isolated microsomal fraction were examined with respect to Ca2+ uptake activity, Ca2+ dependence of phosphorylation from ATP, and the kinetic properties of the phosphoenzyme intermediates formed from both ATP and Pi. The enzymatic cycle was little affected by substitution of Pro160, Pro195, and Pro812, which are located in the cytoplasmic domain; but replacement of Pro308, Pro312, and Pro803, in the putative transmembrane helices, had a profound impact on the function of the enzyme. All mutations of Pro308 and Pro803 led to ATPases which were characterized by a reduced affinity for Ca2+. These prolines may therefore be involved in the structure of the high affinity Ca2(+)-binding sites in the enzyme. Substitution of Pro312 with alanine or glycine gave rise to mutants unable to transport Ca2+ even though their apparent affinities for Ca2+ in the phosphorylation reaction with ATP were increased. In these enzymes, the ADP-sensitive phosphoenzyme intermediate was stable for at least 5 min at 0 degrees C, whereas the ADP-insensitive phosphoenzyme intermediate decay at a rate similar to that of the wild type. Thus, the inability to transport Ca2+ could be accounted for by a block of ADP-sensitive to ADP-insensitive phosphoenzyme intermediate conformational transition. In contrast, substitution of Pro312 with leucine gave rise to a mutant enzyme that retained about 7% of the normal Ca2+ transport rate. Phosphoenzyme turnover in this mutant also occurred at a low but significant rate, suggesting that the leucine side chain can substitute to some extent for proline.
采用位点特异性诱变来研究Pro160、Pro195、Pro308、Pro312、Pro803和Pro812在肌浆网Ca2(+)-ATP酶功能中是否起关键作用。所有六个脯氨酸均被丙氨酸取代；此外，Pro308被甘氨酸取代，Pro312被甘氨酸以及亮氨酸取代。突变cDNA在COS-1细胞中表达，针对分离的微粒体部分中的突变Ca2(+)-ATP酶，检测其Ca2+摄取活性、ATP磷酸化的Ca2+依赖性以及由ATP和Pi形成的磷酸酶中间体的动力学特性。位于胞质结构域的Pro160、Pro195和Pro812的取代对酶促循环影响很小；但位于假定跨膜螺旋中的Pro308、Pro312和Pro803的取代对酶的功能有深远影响。Pro308和Pro803的所有突变均导致ATP酶对Ca2+的亲和力降低。因此，这些脯氨酸可能参与了酶中高亲和力Ca2(+)-结合位点的结构。用丙氨酸或甘氨酸取代Pro312产生了无法转运Ca2+的突变体，尽管它们在与ATP磷酸化反应中对Ca2+的表观亲和力增加。在这些酶中，ADP敏感的磷酸酶中间体在0℃至少稳定5分钟，而ADP不敏感的磷酸酶中间体以与野生型相似的速率衰减。因此，无法转运Ca2+可能是由于ADP敏感向ADP不敏感的磷酸酶中间体构象转变受阻。相比之下，用亮氨酸取代Pro312产生了一种突变酶，其保留了约7%的正常Ca2+转运速率。该突变体中的磷酸酶周转也以低但显著的速率发生，表明亮氨酸侧链可以在一定程度上替代脯氨酸。
