Key analytical aspects of steady-state kinetics
by Dr. Robert Duff, biochemistry manager
Enzymes are mostly proteins which act on a substrate producing product(s). An enzyme functions as a catalyst in a chemical reaction, lowering the activation energy. There are many different types of enzymes: oxidases, catalases, reductases, proteases, nucleases, and isomerases.
Each enzyme will have kinetic parameters that are defined as its intrinsic characteristics with respect to apparent binding affinity (KM), maximum velocity (Vmax) and the maximum “turnover” rate as related to a unique substrate (kcat). The relationship between the KM and Vmax are defined by the Michaelis-Menten equation. Linearization of this equation is represented by the Lineweaver-Burk, and the Eadie- Hofstee plot, where the kinetic parameters are found in the slope of the line (KM) and the Y-intercept (Vmax). The velocity usually follows saturation kinetics with respect to the concentration of substrate. At very high substrate concentrations the reaction rate approaches the maximum velocity.
Enzymatic activity is defined as the measure the catalytic ability of the enzyme. The compendia-based procedures (USP, EP) describe many assays using direct spectrophotometric analysis of activity. While these methods are validated for use, it is usually required that some feasibility be performed to harmonize the method with existing lab equipment. Spectrophotometers such as Molecular Devices M5e MultiMode microwell plate-readers with SoftMax Pro v5.4 (GMP/Part 11 Compliant) can be used for most activity and kinetic assays. The non-linear regression analysis is performed by third party software.
Characterization of enzymes as therapeutics requires careful consideration as to their clinical phase development. In addition to testing for purity and identity, it is important to assess the activity and kinetic parameters of the therapeutic enzyme. With the wide range of techniques possible to aid the analytical scientist today and the advent of newer techniques, determination of these parameters should be straightforward. Assays that were developed and validated in the past are being challenged by newer more sensitive techniques.