Hexokinase equilibrium measurements

As discussed above, an enzymatic reaction is usually found to be more rapid in one direction than the other so that the reaction is virtually irreversible.If the product of the reaction in one direction is removed as it is formed (Le., because it is the substrate of a second enzyme present in the reaction mixture), the equilibrium of the first enzymatic process is displaced so that the reaction may continue to completion in that direction. Reaction sequences in which the product of one enzyme-catalyzed reaction becomes the substrate of another enzyme, often through many stages, are characteristic of metabolic processes. Analytically, several enzymatic reactions also may be Unked together to provide a means of measuring the activity of the first enzyme or the concentration of the initial substrate in the chain. For example, the activity of creatine kinase is usually measured by a series of linked reactions, and glucose can be determined by consecutive reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase. 

Conditions have been established for the measurement of D-glucose using the hexokinase-D-glucose 6-phosphate dehydrogenase method. This has enabled a new approach to be made in kinetic analyses in which the dependencies upon experimental variables are much closer to equilibrium methods than to the more common kinetic methods. 

All the models discussed in this chapter have been essentially equilibrium models that can be applied to kinetic experiments only by assuming that Uo/Finax is a true measure of Fp. However, cooperativity can also arise for purely Idnetic reasons in mechanisms that would show no cooperativity if binding could be measured at equihbrium (Ricard etal., 1966,1974 Ricard Noat, 1984,1986). Examples for such models are rarely described in the literature, and the kinetic model for hexokinase-D represents an example (Neet, 1995). 

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