21] Use of coenzyme analogs to study thiamine diphosphate (cocar☐ylase) binding in yeast pyruvate decar☐ylase (2-Oxoacid car☐y-lyase, EC 4.1.1.1)

Abstract

Publisher Summary This chapter discusses the use of coenzyme analogs to study thiamine diphosphate (cocarboxylase) binding in yeast pyruvate decarboxylase. Schellenberger and co-workers have recently shown that thiamine diphosphate and Mg(II) ions are bound independently to different sites on apopyruvate decarboxylase but that all three components must react together to form the stable holoenzyme complex. With Mg(II) in excess, the Km and Ki values for the coenzyme and its analogs, respectively, may be considered to represent dissociation constants for the apoenzyme–coenzyme and apoenzyme–analog complexes—that is, the relative affinities of the coenzyme and its analogs for the apoenzyme. Because pyruvate decarboxylase activity depends upon the amount of the holoenzyme present, reconstitution of the holoenzyme in the presence of the coenzyme and its analog indicates the extent of interference of the analog with coenzyme binding. The relative affinities of the coenzyme and its analogs for the apoenzyme may then be correlated with the structural features of the molecules involved. The apoenzyme is, thus, preincubated with appropriate concentrations of coenzyme and analog prior to the determination of the enzyme activity.