Watt G D
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Anal Biochem. 1990 May 15;187(1):141-6. doi: 10.1016/0003-2697(90)90432-9.
The mechanism of nitrogenase catalysis, as evaluated from steady-state kinetic measurements, is presently unresolved primarily due to conflicting results regarding the reaction order of the nitrogenase reductant, S2O2-4, at high concentrations. A microcalorimetric method was developed and is described which measures the rate of heat production (and hence the rate of reactant disappearance or product formation) as a function of time. Because each substrate reaction order has a unique profile for the rate of heat production with time, the described procedure provides a means for establishing the substrate reaction order for the enzyme-catalyzed reaction under consideration by visual inspection of the resulting thermogram. The rate constant and other kinetic parameters are obtained from analysis of the shape of the thermogram and thermodynamic parameters are evaluated from either the shape of or the area bound by the thermogram. Application of this procedure to the nitrogenase system has confirmed one-half- and first-order reaction orders under limiting conditions for the S2O2-4 and MgATP substrates during the enzyme-catalyzed reaction for this important biological process. From a single thermogram, the enthalpy of reaction and the kinetic rate law are readily evaluated. The procedure is completely general in nature and is applicable to any chemical or biochemical system that evolves heat.
从稳态动力学测量评估的固氮酶催化机制，目前主要由于在高浓度下关于固氮酶还原剂连二亚硫酸根（S2O2-4）反应级数的结果相互矛盾而未得到解决。本文开发并描述了一种微量量热法，该方法测量产热速率（从而反应物消失速率或产物形成速率）随时间的变化。由于每种底物反应级数对于产热速率随时间的变化都有独特的曲线，所描述的程序提供了一种通过目视检查所得热谱图来确定所考虑的酶催化反应底物反应级数的方法。速率常数和其他动力学参数通过对热谱图形状的分析获得，热力学参数则从热谱图的形状或热谱图所界定的面积进行评估。将该程序应用于固氮酶系统，已证实在此重要生物过程的酶催化反应中，在连二亚硫酸根（S2O2-4）和MgATP底物的极限条件下，反应级数分别为一级半和一级。从单个热谱图中，可以很容易地评估反应焓和动力学速率定律。该程序本质上是完全通用的，适用于任何产生热量的化学或生化系统。
