Reversible folding of UDP-galactose 4-epimerase from Escherichia coli.
UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39-kDa subunits having 1 or 2 molecules of NAD bound non-covalently/dimer. The enzyme can be dissociated and denatured by 8 M urea at pH 7.0 to a state having only 15% of residual secondary structure. Dilution of the denaturant by 20 mM potassium phosphate, pH 8.5, leads to functional reconstitution of the enzyme. No addition of extraneous NAD is required for reactivation, indicating a strong affinity of the cofactor for refolded molecule. The reactivation follows a second-order kinetics (k = 1.2 +/- 0.07 X 10(3) M(-1) s(-1) at 25 degrees C) with an energy of activation of 23.79 +/- 0.33 kJ/mol. The native, denatured and renatured states of the enzyme were characterized by far-ultraviolet CD spectra for secondary structure: protein fluorescence, interaction with extrinsic fluorescence probe ANS (1-anilino 8-naphthalene sulfonic acid) and ultraviolet absorption spectra for tertiary structure and size-exclusion HPLC, gel-filtration chromatography and light-scattering for quaternary structure. The folding process could be broadly divided into two distinct steps: (a) regain of secondary structure and dimerization were fast and were complete within 2 min and 9 min, respectively, and (b) regain of catalytic activity was slow and was complete by 45 min. No active holoenzyme could be identified. It appears that generation of the NAD-binding site and subsequent assembly of NAD is the rate-limiting step expressing catalytic activity.
Dutta
S
S
Indian Institute of Chemical Biology, Calcutta.
Maiti
N R
NR
Bhattacharyya
D
D
D001665:Binding Sites; D002942:Circular Dichroism; D019281:Dimerization; D004793:Enzyme Reactivators / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D004793:Enzyme Reactivators; D009243:NAD; D014534:UDPglucose 4-Epimerase