Voskoboyev A I, Artsukevich I M, Ostrovsky Y M
Acta Vitaminol Enzymol. 1983;5(2):105-13.
The functional role of histidine and sulfhydryl groups of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, E.C.2.7.6.2) has been studied by the methods of photooxidation and chemical modification by diethylpyrocarbonate (DEPC) and Ellman's reagent, 5,5' - dithiobis (2 - nitrobenzoic acid) (DTNB). Histidine amino acid residues have been shown to be destroyed during photoinactivation. Titration of the protein with DEPC has established that modification of two histidine groups decreases the catalytic activity of thiamine pyrophosphokinase. Interaction of the subsequent three groups with the reagent does not affect residual activity. Substrates protect thiamine pyrophosphokinase from inactivation. Two -SH groups have been identified in a molecule of thiamine pyrophosphokinase with Ellman's reagent. Modification of only one of them results in complete loss of the enzymatic activity. Treatment of the enzyme with 8M urea has shown no differences in the amount of thiol groups of the native and denatured enzymes. Mg2+ + ATP and somewhat more weakly Mg2+ partially protect thiamine pyrophosphokinase from inhibition by DTNB. In their presence a high degree of enzyme reactivation is observed. Both histidine and sulfhydryl groups are suggested to play an important role in the catalytic mechanism of thiamine pyrophosphokinase.
通过光氧化以及焦碳酸二乙酯（DEPC）和埃尔曼试剂5,5'-二硫代双（2-硝基苯甲酸）（DTNB）化学修饰的方法，对硫胺素焦磷酸激酶（ATP：硫胺素焦磷酸转移酶，E.C.2.7.6.2）中组氨酸和巯基的功能作用进行了研究。已证明在光灭活过程中组氨酸氨基酸残基会被破坏。用DEPC对该蛋白进行滴定确定，两个组氨酸基团的修饰会降低硫胺素焦磷酸激酶的催化活性。随后三个基团与该试剂的相互作用并不影响残余活性。底物可保护硫胺素焦磷酸激酶不被灭活。用埃尔曼试剂已鉴定出硫胺素焦磷酸激酶分子中有两个巯基。仅其中一个巯基被修饰就会导致酶活性完全丧失。用8M尿素处理该酶后发现，天然酶和变性酶的巯基数量没有差异。Mg2+ + ATP以及较弱程度的Mg2+可部分保护硫胺素焦磷酸激酶不被DTNB抑制。在它们存在的情况下，可观察到酶的高度再活化。组氨酸和巯基均被认为在硫胺素焦磷酸激酶的催化机制中发挥重要作用。
