A microcalorimetric procedure for evaluating the kinetic parameters of enzyme-catalyzed reactions: kinetic measurements of the nitrogenase system.
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.
Watt
G D
GD
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
D001395:Azotobacter / Q000201:enzymology; D002151:Calorimetry / Q000379:methods*; D002384:Catalysis; D007700:Kinetics; D009591:Nitrogenase / Q000378:metabolism*
D009591:Nitrogenase