Chin D, Means A R
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1996 Nov 29;271(48):30465-71. doi: 10.1074/jbc.271.48.30465.
The 9 methionine residues of vertebrate calmodulin (CaM) were individually changed to glutamine residues in order to investigate their roles in enzyme binding and activation. The mutant proteins showed three classes of effect on the activation of smooth muscle myosin light chain kinase, CaM-dependent protein kinase IIalpha, and CaM-dependent protein kinase IV. First, some mutations had no appreciable effect on the ability of CaM to activate the three protein kinases. Included in this category were glutamine substitutions at residues 36 and 51 in the N-terminal domain, at residue 76 in the domain linker sequence, and at residues 144 and 145 in the C-terminal domain. Second, glutamine substitutions in the N-terminal domain of CaM, particularly those at positions 71 and 72, lowered the maximal activity of smooth muscle myosin light chain kinase while having no effect on the other two enzymes. Finally the affinity of CaM for all three enzymes was lowered by glutamine mutations at the neighboring methionines 109 and 124, located on a solvent-accessible surface of the C-terminal domain of Ca2+/CaM. This last result provides the first demonstration of the involvement of the same hydrophobic groups in the high affinity binding of CaM to three different enzymes.
为了研究其在酶结合和激活中的作用，将脊椎动物钙调蛋白（CaM）的9个甲硫氨酸残基分别替换为谷氨酰胺残基。突变蛋白对平滑肌肌球蛋白轻链激酶、CaM依赖性蛋白激酶IIα和CaM依赖性蛋白激酶IV的激活表现出三类效应。首先，一些突变对CaM激活这三种蛋白激酶的能力没有明显影响。这一类包括N端结构域中第36和51位残基、结构域连接序列中第76位残基以及C端结构域中第144和145位残基的谷氨酰胺替代。其次，CaM N端结构域中的谷氨酰胺替代，特别是第71和72位的替代，降低了平滑肌肌球蛋白轻链激酶的最大活性，而对其他两种酶没有影响。最后，位于Ca2+/CaM C端结构域溶剂可及表面的相邻甲硫氨酸109和124处的谷氨酰胺突变降低了CaM对所有三种酶的亲和力。最后这一结果首次证明了相同的疏水基团参与了CaM与三种不同酶的高亲和力结合。
