The Michaelis constant has been found to decrease by more than one order of magnitude when substrate of opposite charge of that of the carrier matrix was used. Again, this only happened at low ionic strengths, and when neutral substrates were used. The electrostatic potential was calculated by insertion of the Maxwell-Boltzmann distribution into the Michaelis-Menten equation using the changes in Michaelis constant. Good agreement was obtained with the value for the electrostatic potential calculated from the pH-activity shifts.
It is recognized that the kinetic constants measured with immobilized enzymes are not true kinetic constants equivalent to those obtained in homogeneous reactions. They are apparent values because of the effects of diffusion and partitioning. Hence, maximum velocity and Michaelis constants should be referred to as apparent Vmax and apparent Km.
The diffusion of substrate from the bulk solution to the micro-environment of an immobilized enzyme can limit the rate of the enzyme reaction. The rate at which substrate passes over the insoluble particle affects the thickness of the diffusion film, which in turn determines the concentration of substrate in the vicinity of the enzyme and hence the rate of reaction.
Molecular weight of the substrate can also play a large role. Diffusion of large molecules will obviously be limited by steric interactions with the matrix. This is reflected in the fact that the relative activity of bound enzymes towards high molecular weight substrates has generally been found to be lower than towards low molecular weight substrates. This, however, may be an advantage in some cases, since the immobilized enzymes may be protected from attack by large inhibitor molecules.
