Peterson Michelle E, Daniel Roy M, Danson Michael J, Eisenthal Robert
Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand.
Biochem J. 2007 Mar 1;402(2):331-7. doi: 10.1042/BJ20061143.
Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by DeltaG(Dagger)(cat), and irreversible inactivation defined by DeltaG(Dagger)(inact). However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (E(inact)) that is in reversible equilibrium with the active form (E(act)); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, DeltaH(eq), and a new thermal parameter, T(eq), which is the temperature at which the concentrations of E(act) and E(inact) are equal; T(eq) may therefore be regarded as the thermal equivalent of K(m). Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of T(eq) and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.
传统上，酶活性对温度的依赖性是由一个包含两个过程的模型来描述的：由ΔG‡(cat)定义的催化反应，以及由ΔG‡(inact)定义的不可逆失活。然而，这样的模型并不能解释所观察到的酶的温度依赖性行为，因此已经开发并验证了一个新的模型。这个模型（平衡模型）描述了一种新的机制，即酶在高温下失活的机制，它包括一种酶的无活性形式（E(inact)），它与活性形式（E(act)）处于可逆平衡状态；正是这种无活性形式经历不可逆的热失活转变为热变性状态。这个平衡由一个平衡常数来描述，其温度依赖性通过平衡焓ΔH(eq)和一个新的热参数T(eq)来表征，T(eq)是E(act)和E(inact)浓度相等时的温度；因此，T(eq)可以被视为Km的热等效物。因此，对一种酶的温度依赖性行为进行表征必须包括对这些内在性质的测定。平衡模型对酶学、生物技术以及理解酶的进化具有重要意义。本研究提出了一种基于直接将进展曲线拟合到平衡模型的新的直接数据拟合方法，并评估了该方法的稳健性以及测定数据对准确测定T(eq)及其相关参数的影响。它还描述了比以前可用的方法更简单的用于测定这些参数的实验方法，包括将平衡模型应用于非理想酶反应所需的方法。
