Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase.
Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase.
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DOI：
10.1021/bi00539a017
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发表时间：
1982-05
期刊：
影响因子：
2.9
通讯作者：
Donald K. Blumenthal;J. Stull
中科院分区：
文献类型：
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作者：
Donald K. Blumenthal;J. Stull
Ca42+-钙调蛋白与肌球蛋白轻链激酶非活性催化亚基的可逆结合导致了具有催化活性的全酶复合体的形成[Blumenthal，D.K.，&Stull，J.T.(1980)生化19,5608-5614]。本研究是为了确定pH、温度和离子强度对活化和催化过程的影响。当肌球蛋白轻链激酶被钙调蛋白完全激活时，表现出较宽的最适pH(在pH 6.5-pH 9.0范围内大于最大活性的90%)，仅被中等离子强度轻微抑制(在Mu=0.22时抑制不到20%)，并表现出明显的温度依赖性(Q10等于2；Ea=10.4kcal mol-1)。根据Arrhenius图计算的热力学参数表明，与催化限速步骤相关的吉布斯能垒主要是热焓。钙调蛋白激活该酶的最适pH范围(pH 6.0~-7.5)比催化活性要窄，并受到离子强度的显著抑制(在Mu=0.22时抑制程度大于70%)，呈现非线性的van‘t Hoff曲线。在10-20℃之间，活化主要是由熵驱动的(S增量为40卡分子-1℃；增量H度为-900卡分子-1)，但在20-30℃之间，焓因素占主导地位(S增量为10卡分子-1度；增量H度为-9980卡分子-1)。活化过程中的表观热容变化为-910卡·摩尔~(-1)·度~(-1)。在这些数据的基础上，我们认为，尽管钙调蛋白和激酶之间的疏水相互作用是酶激活所必需的，但其他类型的相互作用，如氢键、离子和范德华相互作用，也对激活过程做出了重要的贡献，可能是必不可少的。
The reversible association of Ca42+-calmodulin with the inactive catalytic subunit of myosin light chain kinase results in the formation of the catalytically active holoenzyme complex [Blumenthal, D. K., & Stull, J. T. (1980) Biochemistry 19, 5608--5614]. The present study was undertaken in order to determine the effects of pH, temperature, and ionic strength on the processes of activation and catalysis. The catalytic activity of myosin light chain kinase, when fully activated by calmodulin, exhibited a broad pH optimum (greater than 90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (less than 20% inhibition at mu = 0.22), and displayed a marked temperature dependence (Q10 congruent to 2; Ea = 10.4 kcal mol-1). Thermodynamic parameters calculated from Arrhenius plots indicate that the Gibb's energy barrier associated with the rate-limiting step of catalysis is primarily enthalpic. The process of kinase activation by calmodulin had a narrower pH optimum (pH 6.0--7.5) than did catalytic activity, was markedly inhibited by increasing ionic strength (greater than 70% inhibition at mu = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 degrees C, activation was primarily entropically driven (delta S degrees congruent to 40 cal mol-1 deg-1; delta H degrees = -900 cal mol-1), but between 20 and 30 degrees C, enthalpic factors predominated in driving the activation process (delta S degrees congruent to 10 cal mol-1 deg-1; delta H degrees = -9980 cal mol-1). The apparent change in heat capacity (delta Cp) accompanying activation was estimated to be -910 cal mol-1 deg-1. On the basis of these data we propose that although hydrophobic interactions between calmodulin and the kinase are necessary for the activation of the enzyme, other types of interactions such as hydrogen bonding, ionic, and van der Waals interactions also make significant and probably obligatory contributions to the activation process.