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Equilibria of reactions catalyzed by acetate kinase and adenylate kinase were studied experimentally and theoretically. Nucleotide conversions in excess of 90% were obtained with reactants (acetyl phosphate and one nucleotide) in stoichiometric proportion for ATP regeneration from either ADP via acetate kinase or AMP via the coupled enzyme system. Observed equilibrium constants, measured as a function of [Mg2+] at pH 7.4, ranged from about 1 to 9 for the reaction catalyzed by adenylate kinase and from about 50 to 400 for the reaction catalyzed by acetate kinase. These results agreed well with prediction of a theoretical model for the multiple equilibria between all species present in solution. Theoretical calculations showed that magnesium ion complexes and totally dissociated anions are the predominant species in solution at .

Lineweaver-Burk plots of reaction rate data obtained with immobilized enzymes need not be linear even when intrinsic enzyme kinetics follow the simple Michaelis-Menten rate expression. Theoretical calculations show that mass transfer effects may cause curvature which is concave or convex to the abscissa, depending upon experimental conditions. Consequently, graphical procedures commonly employed for analysis of soluble enzyme kinetics may yield misleading results when applied to immobilized enzymes. Three approaches which follow from the behavior of numerical and asymptotic solutions to the problem are proposed for extraction of intrinsic kinetic information.

Process engineering considerations important in the exploitation of enzyme-catalyzed reactions for large-scale production of desired products are illustrated in the context of a case study of glucose isomerase technology. The state of the art of glucose isomerase processing as revealed by journal and patent literature is reviewed and assessed. Among topics covered are enzyme production, immobilization and stabilization, kinetics, reactor design, and product recovery. Some possible future processing objectives, such as production of pure fructose, are discussed.

The observed kinetics of immobilized enzymes can often be influenced by mass transfer effects. To ensure straight-forward and useful analysis of data, these influences should be anticipated at the early stages of design and execution of kinetic experiments. With adequate care, it is then possible to interpret meaningfully values of commonly reported immobilized enzyme kinetic parameters, such as the “apparent Michaelis constant,” even if severe mass transfer effects are present when rate data are obtained.

Shuler, Aris & Tsuchiya (1972) have recently considered the combined effects of diffusive resistance and electrostatic field on the rate of reaction catalyzed by an enzyme immobilized on a non-porous surface. They employed a potential distribution for the electrical double layer which is asymptotically valid when surface potential is small.The complete Gouy-Chapman solution, which is valid for higher surface potential, is employed here. Numerical values of the effectiveness factor calculated with this potential distribution agree very closely with the results of Shuler et al. for most cases. It is shown that the effectiveness factor can (i) attain magnitudes much greater than unity in physically realizable systems, (ii) approach the solution for “infinite” surface potential at reasonable values of surface charge density, and (iii) pass through a maximum as bulk substrate concentration is varied. This behavior leads to the existence of an optimum surface concentration for enzyme immobilized on a highly charged non-porous support such that the most effective catalytic action on a charged substrate is ensured. Finally, it is established that significant electrical and/or diffusive effects result in non-linear Lineweaver-Burk plots of reciprocal observed reaction velocity against reciprocal bulk substrate concentration. These non-linear plots cannot be interpreted in the same way as linear plots obtained when enzyme is unbound.

The authors describe a simple model reaction system in order to illustrate how selective catalysts can affect practical equilibrium concentrations and yields.