Evaluation of optimum conditions of two-substrate enzyme reactions.

A modern approach is described for the evaluation of the optimal conditions for two-substrate enzyme reactions. It chiefly involves the determination of the concentration of substrates for the primary reaction and the catalytic concentration of indicator enzymes. The interrelationship between the concentration of the two substrates (concentration pairs) are described mathematically to be hyperbolic, and, in case of competitively inhibited reactions, to be parabolic. Calculated optimum concentrations have been rechecked experimentally for the reactions of aspartate aminotransferase and alanine aminotransferase. For pyridine coenzyme linked indicator reactions it could be demonstrated that they mostly follow zero order kinetics. One of the products of the primary reaction reacts, in its steady state concentration, as the second substrate. This represents the size of the lag phase of the coupled reaction. The Km of this substance must be known in order to calculate the catalytic concentration of the indicator enzyme in relation to that of the primary enzyme. Its concentration can be fixed arbitrarily within certain limits, depending on whether the calculated result actually can be realized; otherwise a larger lag phase must be tolerated. For practical reasons, it is generally possible to measure only a certain percentage of maximum reaction rate.