Theory and practical application of coupled enzyme systems: one and two coupling enzymes with mutarotation of an intermediate.

This paper provides equations to calculate the elapsed time before the concentration of the final intermediate, in a sequence of coupled enzymatic reactions, achieves a defined fraction of its steady-state concentration when one of the intermediates undergoes mutarotation. The equations can be used to predict lag times for systems involving one coupling enzyme, as is the case when hexokinase or phosphoglucomutase activity is monitored using glucose-6-phosphate dehydrogenase as the auxiliary enzyme, or for systems of two coupling enzymes, as is the case when the activities of enzymes producing ATP (such as creatine kinase) are monitored by coupling the production of ATP to hexokinase and glucose-6-phosphate dehydrogenase. The theoretical aspects of the assay have been verified using hexokinase (as the primary enzyme) and glucose-6-phosphate dehydrogenase (as the coupling enzyme). A method of cost minimization, based on the above relationships, is also provided.