Abstract
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.
Highlights
This epimerase converts both the substrates, UDPglucose association of coenzymeNAD to the apoenzyme
For some of the classical dehydrogenases, a ence of theessential histidyl residue in close proximity histidyl residue seems to function as theprototropic catalyst to the coenzyme binding regionof the active site
Earlier work inourlaboratory on the reconstituted yeast enzyme system withCibacron blue [10] and with ethenoNAD [11] indicated that significant tertiary structurehomology at thepyridine nucleotide binding region of the active site exists between this epimerase and the dehydrogenases
Summary
Diethylpyrocarbonate was the product of Aldrich Chemical Co. Other biochemicals were purchased from Sigma. The enzyme activity was assayed spectrophotometrically according to the coupled assay procedure of Darrow and Rodstrom [4] or by a twostep assay method described previously [16]. Stock concentrationof diethylpyrocarbonate was determined spectrophotometrically by reaction with 20 mM L-histidine in 0.02 M potassium phosphate buffer, pH 7.0, and measuring the increase in absorbance at 230 nm (e = 3000 M" cm") [14]. The final concentration of ethanol in the reaction mixture ranged from 1% to 10% by volume and was found to have no significant effect on the activity and stability of the enzyme during the incubation time. The value of k' (first order rate constant for hydrolysis of diethylpyrocarbonate) was obtained by measuring the amountof diethylpyrocarbonate remaining at various times on treatment with 20 mM histidine in 0.02 M potassium phosphate buffer, pH 7.0, as described above. Plot of log (pseudo-first order rate constants for inactivation (&bs)) obtained at various concentrations of diethylpyrocarbonate against log concentration of the reagent
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