UDP-GALACTOSE 4-EPIMERASE FROM KLUYVEROMYCES-FRAGILIS - RECONSTITUTION OF HOLOENZYME STRUCTURE AFTER DISSOCIATION WITH PARACHLOROMERCURIBENZOATE

UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis (Kluyverom yces marxianus var. marxianus) is a homodimer of molecular mass 75 kDa /subunit and has one mol NAD firmly bound/dimer. The pathway for the a ssembly of the holoenzyme structure has been studied after dissociatin g the native epimerase with p-chloromercuribenzoate into inactive merc urated monomers. The process of dissociation was not associated with u nfolding of the molecules. Reconstitution of the functional holoenzyme was done by reduction with dithiothreitol and addition of extra NAD. The reaction was thus followed to monitor maturation of the enzyme fro m the folded monomeric state. The reconstituted enzyme was similar to the native enzyme in terms of a number of physiochemical properties su ch as secondary, tertiary and quarternary structures, K-m for the subs trate UDP-galactose, reductive inhibition, interaction with the fluoro phore 1-anilino 8-naphthalene sulphonic acid (ANS), etc. Reconstitutio n under low ionic strength buffer (I = 0.011) shows that the presence of NAD is essential for the formation of a dimeric structure. However, dimeric apoenzyme could also be stabilized under high ionic strength buffer (I = 0.1). Reactivation was strongly dependent on pH, being mos t effective at pH 8.1. Kinetic evidence suggested that, at low ionic s trength, assembly of NAD over dimeric apoenzyme is the rate-limiting s tep in expressing catalytic activity. This process has a low energy of activation of 27.2 kJ/mol.