Gao Z H, Krebs J, VanBerkum M F, Tang W J, Maune J F, Means A R, Stull J T, Beckingham K
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1993 Sep 25;268(27):20096-104.
Activation of four target enzymes by two series of calmodulin Ca2+ binding site mutants has been examined. In each mutant, the conserved bidentate glutamate of one of the Ca2+ binding sites is mutated to glutamine or lysine. The enzymes studied were smooth and skeletal muscle myosin light chain kinases, adenylylcyclase, and plasma membrane Ca(2+)-ATPase. For the first three enzymes, the activation patterns with the two mutant series were very similar: mutation of site 4 was most deleterious, then site 2, site 3, and site 1. This ranking was observed previously in Ca2+ binding and Ca(2+)-induced conformational studies of these mutants. Thus the response of these enzymes is probably determined by the extent to which each mutant's competence to interact with target binding regions has been compromised. In contrast, for Ca(2+)-ATPase, mutants of sites 3 and 4 were much poorer activators than those of sites 1 and 2. Events beyond calmodulin binding and related to enzyme activation probably dictate this unusual activation pattern and also the anomalously poor activation of skeletal muscle myosin light chain kinase by site 1 mutant B1Q. Site 1 mutant B1K showed wild type activation of all four enzymes suggesting that in site 1, the lysine substitution can evoke the conformational changes associated with Ca2+ binding.
研究了两系列钙调蛋白Ca2+结合位点突变体对四种靶酶的激活作用。在每个突变体中，其中一个Ca2+结合位点保守的双齿谷氨酸被突变为谷氨酰胺或赖氨酸。所研究的酶有平滑肌和骨骼肌肌球蛋白轻链激酶、腺苷酸环化酶以及质膜Ca(2+)-ATP酶。对于前三种酶，两个突变体系的激活模式非常相似：位点4的突变最有害，其次是位点2、位点3和位点1。这种排序先前在这些突变体的Ca2+结合和Ca(2+)诱导的构象研究中也观察到。因此，这些酶的反应可能取决于每个突变体与靶结合区域相互作用能力受损的程度。相比之下，对于Ca(2+)-ATP酶，位点3和4的突变体作为激活剂比位点1和2的突变体差得多。钙调蛋白结合之外与酶激活相关的事件可能决定了这种不寻常的激活模式，也决定了位点1突变体B1Q对骨骼肌肌球蛋白轻链激酶异常差的激活作用。位点1突变体B1K对所有四种酶均表现出野生型激活，这表明在位点1中，赖氨酸取代可引发与Ca2+结合相关的构象变化。
