Acyl phosphate and cyclic AMP inhibition of reactions of D-glyceraldehyde-3-phosphate dehydrogenase with aldehyde and acyl phosphate substrates: multiple inhibition analysis.

B R DeMark, B M Benjamin, T H Fife

Index: Arch. Biochem. Biophys. 223(2) , 360-8, (1983)

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Abstract

The effects of the inhibitors trimethylacetyl phosphate and cAMP have been determined in reactions catalyzed by D-glyceraldehyde-3-phosphate dehydrogenase. These inhibitors must influence the oxidation of aldehydes through substrate dependent cooperative conformational changes. Both trimethylacetyl phosphate and cAMP give sigmoidal 1/V vs (I) plots in oxidation of glyceraldehyde 3-phosphate, but exert linear competitive effects on the acyl phosphatase site in acylation reactions of beta-(2-furyl) acryloyl phosphate. The linear inhibition in the latter reactions indicates that one inhibitor molecule is bound per active site. Hydride transfer to NAD+ is the rate-determining step in oxidation of benzaldehyde to an acylenzyme, as shown by the threefold decrease in Vmax without change in Km when 1-deuterobenzaldehyde is the substrate; it is very likely this step that is affected by acyl phosphate inhibitors. Plots of 1/V vs cAMP concentration for oxidation of benzaldehyde at a series of trimethylacetyl phosphate concentrations are parallel at concentrations of acyl phosphate less than 0.00625 M, which demonstrates that binding of the inhibitors is mutually exclusive. However, at higher concentrations of trimethylacetyl phosphate, the slopes are affected, which shows that both inhibitors are then binding. Thus, the binding of high concentrations of acyl phosphate must result in a conformational change of the enzyme that permits binding of both inhibitors. A number of conformations with different kinetic properties are formed with the various substrate and inhibitor combinations. In reactions of muscle D-glyceraldehyde-3-phosphate dehydrogenase, binding of these inhibitors is best explained in terms of induced fit and a sequential model of conformational changes.