title: UDP-galactose 4-epimerase from Kluyveromyces fragilis: existence of subunit independent functional site.
abstract: UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with non-covalently bound NAD acting as cofactor. Partial proteolysis with trypsin in the presence of 5'-UMP, a strong competitive inhibitor, led to a degraded product which was purified. Results from SDS-PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa. 'Two-step assay' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer. The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD. This truncated enzyme retained catalytic site related properties like Km for UDP-galactose, 'NADH-like coenzyme fluorescence' and 'reductive inhibition' similar to its dimeric counterpart. Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure. Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer. Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved. Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a 'subunit sharing model' of its catalytic site.
authors.affiliation: Division of Drug Design, Development and Molecular Modeling, Indian Institute of Chemical Biology, 4, Raja S.C. Mallick Road, Jadavpur, Calcutta 700032, India.
authors.forename: Amrita
authors.initials: A
authors.lastname: Brahma
authors.forename: Debasish
authors.initials: D
authors.lastname: Bhattacharyya
mesh_terms: D002384:Catalysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007716:Kluyveromyces / Q000201:enzymology*; D008970:Molecular Weight; D014534:UDPglucose 4-Epimerase / Q000302:isolation & purification / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D014534:UDPglucose 4-Epimerase