Wang Y, Wong S S, Fukuda M N, Zu H, Liu Z, Tang Q, Appert H E
Department of Surgery, Medical College of Ohio, Toledo 43699.
Biochem Biophys Res Commun. 1994 Oct 28;204(2):701-9. doi: 10.1006/bbrc.1994.2516.
The functions of the five cysteine residues in human galactosyltransferase were investigated using site-directed mutagenesis to determine the location of the disulfide bond as well as the role of the sulfhydryl groups. The enzyme remains active when three of its cysteine residues at positions 171, 264 and 340 are mutated to serine separately. However, enzymatic activity is lost when either cysteine-129 or cysteine-245 is replaced with serine. The loss of GT activity suggests that these two cysteine residues form a disulfide bond. The three active mutated enzymes were studied kinetically. The kinetic constants of the enzymes with cysteine-171 or cysteine-264 replaced with serine are not significantly different from those of GT that does not have these substitutions. When cysteine-340 was mutated, however, the kinetic constant for UDP-galactose increased about 30 fold, while that for N-acetylglucosamine and Mn2+ remained unchanged. In addition, sulfhydryl inhibition studies reveal that cysteine-340 is the only cysteine residue that reacts with the sulfhydryl reagents. These results indicate that cysteine-340 may be involved in the binding of UDP-galactose.
利用定点诱变研究了人半乳糖基转移酶中五个半胱氨酸残基的功能，以确定二硫键的位置以及巯基的作用。当该酶位于171、264和340位的三个半胱氨酸残基分别突变为丝氨酸时，酶仍保持活性。然而，当半胱氨酸-129或半胱氨酸-245被丝氨酸取代时，酶活性丧失。GT活性的丧失表明这两个半胱氨酸残基形成了一个二硫键。对三种有活性的突变酶进行了动力学研究。半胱氨酸-171或半胱氨酸-264被丝氨酸取代的酶的动力学常数与未进行这些取代的GT的动力学常数没有显著差异。然而，当半胱氨酸-340发生突变时，UDP-半乳糖的动力学常数增加了约30倍，而N-乙酰葡糖胺和Mn2+的动力学常数保持不变。此外，巯基抑制研究表明，半胱氨酸-340是唯一与巯基试剂反应的半胱氨酸残基。这些结果表明，半胱氨酸-340可能参与UDP-半乳糖的结合。
