Enzyme Kinetics on pH

Another frequently used method for pK determination is based on measurements of the pH dependence of enzyme kinetics. The pH influences the kinetics of the enzymatic reaction in three ways. It affects maximal velocity, the formation of the enzyme-substrate complex, and the stability of the protein. 

For the sake of completeness, let us start by recalling some basic equations governing enzyme kinetics. A simple mechanism consistent with experimentally observed kinetic data is the Michaelis-Menten relation °  

Studies of enzyme kinetics involve measurements of initial velocity v of the reaction as a function of substrate concentration [S]. Values of the kinetic constants are determined by fitting the initial velocity and concentration data to the appropriate rate equations by the least-squares method. The maximal velocity and Michaelis constant are obtained from experimental data, usually from different methods of plotting the kinetic parameters, the most common of which is to plot llv versus 

This plot, the Lineweaver-Burk or double-reciprocal plot, gives a strong weighting to results at low substrate concentrations and should not be used without proper weighting statistics. Another form of the plot is 

This is a highly simplified kinetic mechanism however, it is sufficient to illustrate the general approach that is usually applied. Here the protons have been omitted for the sake of simplicity. Because proteolytic reactions are usually very fast (especially in the presence of buffers, where proton transfer reactions occur readily), all of the proteolytic steps can be assumed to be in equilibrium throughout the course of the reaction. and represent the molecular dissociation constants for the free enzyme, and and represent those for 

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