Abstract
UDP-glucose 4-epimerase from Saccharomyces fragilis has 1 mol of NAD firmly bound per mol of the dimeric apoenzyme. This prevents a direct study of the coenzyme binding site of the protein. Dissociation of the dimer with p-chloromercuribenzoate and its reconstitution with exogenous NAD or one of its analogues and 2-mercaptoethanol provides an indirect method of study of the site. Depending on the reconstitution properties, the analogues can be classified in the following groups: (i) analogues that have no affinity for the site; (ii) analogues that have affinity but are not incorporated into the apoenzyme; (iii) analogues that produce catalytically inactive holoenzymes; and (iv) analogues that produce catalytically active holoenzymes. Minimum structural requirements that lead to affinity for the coenzyme site and to binding to the apoenzyme can also be discerned from these studies. Reconstitution with etheno-NAD, a fluorescent analogue of NAD, indicates the presence of a hydrophobic pocket for the adenosine subsite.
Published Version
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