UDP-galactose 4-epimerase from Kluyveromyces fragilis: reconstitution of holoenzyme structure after dissociation with parachloromercuribenzoate.
UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis (Kluyveromyces marxianus var. marxianus) is a homodimer of molecular mass 75 kDa/subunit and has one mol NAD firmly bound/dimer. The pathway for the assembly of the holoenzyme structure has been studied after dissociating the native epimerase with p-chloromercuribenzoate into inactive mercurated monomers. The process of dissociation was not associated with unfolding 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 from the folded monomeric state. The reconstituted enzyme was similar to the native enzyme in terms of a number of physiochemical properties such as secondary, tertiary and quarternary structures, Km for the substrate UDP-galactose, reductive inhibition, interaction with the fluorophore 1-anilino 8-naphthalene sulphonic acid (ANS), etc. Reconstitution 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 most effective at pH 8.1. Kinetic evidence suggested that, at low ionic strength, assembly of NAD over dimeric apoenzyme is the rate-limiting step in expressing catalytic activity. This process has a low energy of activation of 27.2 kJ/mol.
Majumdar
S
S
Division of Protein Engineering, Indian Institute of Chemical Biology, Calcutta.
Bhattacharjee
H
H
Bhattacharyya
D
D
Bhaduri
A
A
D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D004789:Enzyme Activation; D020035:Holoenzymes / Q000737:chemistry*; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D009994:Osmolar Concentration; D011487:Protein Conformation; D014534:UDPglucose 4-Epimerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D020035:Holoenzymes; C027132:1-anilino-8-naphthalenesulfonate; D014534:UDPglucose 4-Epimerase