Nishimura H, Sempuku K, Iwashima A
Biochim Biophys Acta. 1981 May 29;668(3):333-8. doi: 10.1016/0005-2795(81)90166-5.
The reaction of a soluble thiamine-binding protein of Saccharomyces cerevisiae with water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, at pH 4.5, results in a remarkable loss of its binding activity with thiamine. Thiamine above 0.1 mM substantially protects the protein against this inactivation. In addition to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the thiamine-binding protein is also inactivated by diethylpyrocarbonate. The inactivation is time-dependent and follows second-order kinetics. Restoration of the binding activity by incubation of inactivated protein with hydroxylamine was observed. thiamine and pyrithiamine are effective to prevent the inactivation. From these results it is strongly suggested that both the carboxyl and the histidine residues in the protein are involved in the binding site for thiamine. It is proposed that the binding involves interactions between charged groups on the protein with the quaternary nitrogen of the thiazolium moiety and with the basic ring nitrogen of the pyrimidine moiety in thiamine molecule.
酿酒酵母的一种可溶性硫胺素结合蛋白在pH 4.5条件下与水溶性碳二亚胺1-乙基-3-(3-二甲基氨基丙基)碳二亚胺反应，会导致其与硫胺素的结合活性显著丧失。浓度高于0.1 mM的硫胺素能有效保护该蛋白不被灭活。除了1-乙基-3-(3-二甲基氨基丙基)碳二亚胺外，硫胺素结合蛋白也会被焦碳酸二乙酯灭活。这种灭活具有时间依赖性，遵循二级动力学。观察到将失活的蛋白与羟胺一起孵育可恢复其结合活性。硫胺素和吡硫胺素可有效防止这种灭活。从这些结果强烈表明，该蛋白中的羧基和组氨酸残基都参与了硫胺素的结合位点。有人提出，这种结合涉及蛋白上的带电基团与硫胺素分子中噻唑鎓部分的季铵氮以及嘧啶部分的碱性环氮之间的相互作用。
