An essential histidine residue for the activity of UDPglucose 4-epimerase from Kluyveromyces fragilis.
UDPglucose 4-epimerase from Kluyveromyces fragilis was completely inactivated by diethylpyrocarbonate following pseudo-first order reaction kinetics. The pH profile of diethylpyrocarbonate inhibition and reversal of inhibition by hydroxylamine suggested specific modification of histidyl residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of 1 essential histidine residue to be responsible for loss in catalytic activity of yeast epimerase. No major structural change in the quarternary structure was observed in the modified enzyme as shown by the identical elution pattern on a calibrated Sephacryl 200 column and association of coenzyme NAD to the apoenzyme. Failure of the substrates to afford any protection against diethylpyrocarbonate inactivation indicated the absence of the essential histidyl residue at the substrate binding region of the active site. Unlike the case of native enzyme, sodium borohydride failed to reduce the pyridine moiety of the coenzyme in the diethylpyrocarbonate-modified enzyme. This indicated the presence of the essential histidyl residue in close proximity to the coenzyme binding region of the active site. The abolition of energy transfer phenomenon between the tryptophan and coenzyme fluorophore on complete inactivation by diethylpyrocarbonate without any loss of protein or coenzyme fluorescence are also added evidences in this direction.
Mukherji
S
S
Indian Institute of Chemical Biology, Calcutta.
Bhaduri
A
A
D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D009243:NAD; D006639:Histidine; D014534:UDPglucose 4-Epimerase; D004047:Diethyl Pyrocarbonate