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 (>90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (<20% inhibition at µ = 0.22), and displayed a marked temperature dependence (Q10 ≈ 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 (>70% inhibition at µ = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 °C, activation was primarily entropically driven ( ∆S° ≈ 40 cal mol-1 deg-1; ∆H° = -900 cal mol-1), but between 20 and 30 °C, enthalpic factors predominated in driving the activation process (∆S° ≈ 10 cal mol-1 deg-1; ∆H° = -9980 cal mol-1). The apparent change in heat capacity (∆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.
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