Reversible folding of UDP-galactose-4-epimerase from yeast Kluyveromyces fragilis.
UDP-galactose-4-epimerase from yeast Kluyveromyces fragilis is a dimeric molecule with one molecule of cofactor NAD per dimer. In presence of 8 M urea, the enzyme is inactivated with complete disorganization of its structure and dissociation of the subunits together with the cofactor. Dilution of the denaturant by sodium phosphate buffer (20 mM, pH 7.0) containing 1 mM NAD recovers the activity to the extent of 80-100%. At a monomer concentration of 0.8 microM, the reactivation follows second-order kinetics, k = 1.48 x 10(3) M-1 s-1, at 20 degrees C. The renatured enzyme resembles the native enzyme in terms of most of its physicochemical properties, e.g., circular dichroism spectrum, fluorescence spectrum, interaction with hydrophobic fluorophore 1-anilino-8-naphthalenesulfonic acid (ANS), hydrodynamic volume, Km for the substrate UDP-galactose, etc. The reactivation process has an energy of activation of 15.2 kcal/mol. The folding pathway could be divided into four major parts: in the first phase (within 10 min), the secondary and tertiary structures of the monomers are formed as found by circular dichroism and fluorescence spectroscopy; in the second phase (within 20 min), the folded monomers associate to form an inactive dimer as observed by size-exclusion HPLC; in the third phase, the NAD binding site is formed, which is possibly induced by the cofactor; and finally, NAD assembles over the cofactor binding site to yield an active holoenzyme (within 120 min). The third and fourth steps have been detected kinetically. They are slow and rate-limiting, depending upon the concentration of extraneous NAD.
Indian Institute of Chemical Biology, Jadavpur, Calcutta.
D
D
Bhattacharyya
D002942:Circular Dichroism; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014534:UDPglucose 4-Epimerase / Q000737:chemistry*
D009243:NAD; D014534:UDPglucose 4-Epimerase