UDP-galactose 4-epimerase from Escherichia coli: formation of catalytic site during reversible folding.
UDP-galactose 4-epimerase from Escherichia coli is a homodimer of molecular weight 39 kDa/subunit having noncovalently bound NAD acting as cofactor. Denaturation by 8 M urea at pH 7.0 causes 85% loss of its secondary structure and dissociation of its constituent molecules. Dilution of the denaturant by buffer at pH 8.5 leads to functional reconstitution of the dimeric holoenzyme. The refolding process is biphasic: after 2 min an equilibrium conformer is formed having 72% of its native secondary structure and by 60 min reactivation becomes complete. The early intermediate has lower energy of activation against thermal denaturation than the reactivated state. Patterns of trypsin digestion suggests a native like structure of this intermediate. Variation of solvent viscosity and ionic strength and inclusion of proline cis-trans isomerase in the refolding process do not alter kinetics of reactivation. Moreover, unaltered kinetics of reactivation against variation of temperature, pH, and duration of denaturation strongly suggests absence of proline cis/trans isomerization. Measurement of kinetics of (i) recovery of tertiary structure by protein fluorescence; (ii) incorporation of NAD from quantitation of bound cofactor; (iii) formation of substrate binding site by specific interaction with extrinsic fluorophore 1-anilino-8-naphthalene sulfonic acid and quenching by 5'-UMP, a competitive inhibitor; and (iv) recovery of activity indicate that they are all comparable. It appears that internal rearrangement of the protein during refolding, shielded from solvent, is the rate-limiting step of generation of cofactor binding site which ultimately leads to maturation of the holoenzyme structure.
Barat
B
B
Indian Institute of Chemical Biology, 4, Raja, S.C. Mullick Road, Jadavpure, Calcutta, 700032, India.
Bhattacharyya
D
D
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014534:UDPglucose 4-Epimerase / Q000737:chemistry* / Q000378:metabolism
D014357:Trypsin; D014534:UDPglucose 4-Epimerase