Mukherji S, Bhaduri A
Indian Institute of Chemical Biology, Calcutta.
J Biol Chem. 1992 Jun 15;267(17):11709-13.
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.
脆壁克鲁维酵母的UDP葡萄糖4-表异构酶在焦碳酸二乙酯作用下按照准一级反应动力学完全失活。焦碳酸二乙酯抑制作用的pH曲线以及羟胺对抑制作用的逆转表明组氨酸残基发生了特异性修饰。对残余酶活性和修饰程度的统计分析表明，1个必需组氨酸残基的修饰导致了酵母表异构酶催化活性的丧失。在校准的Sephacryl 200柱上相同的洗脱模式以及辅酶NAD与脱辅基酶的结合表明，修饰后的酶在四级结构上没有发生重大变化。底物不能提供任何针对焦碳酸二乙酯失活的保护作用，这表明活性位点的底物结合区域不存在必需的组氨酸残基。与天然酶的情况不同，硼氢化钠不能还原焦碳酸二乙酯修饰的酶中辅酶的吡啶部分。这表明在活性位点的辅酶结合区域附近存在必需的组氨酸残基。焦碳酸二乙酯完全失活时色氨酸与辅酶荧光团之间能量转移现象的消除，且蛋白质或辅酶荧光没有任何损失，这也为此提供了更多证据。
