Ohnoki S, Hong B S, Buchanan J M
Biochemistry. 1977 Mar 22;16(6):1065-9. doi: 10.1021/bi00625a006.
A two-step method for labeling the glutamine active site of formyglycinamide ribonucleotide (FGAR) amidotransferase from chicken liver has been developed in which reaction of all other reactive groups with unlabeled iodoacetate is followed by specific labeling of the glutamine site with radioactive reagent. A study of the reaction as a function of duration, temperature, and pH of the incubation as well as concentration of iodoacetate has revealed that two nonessential groups of the enzyme react in the presence of glutamine and that this modified enzyme is relatively resistant to further carboxymethylation. When this modified enzyme was incubated with radioactive iodoacetate in the presence of FGAR, ATP, and Mg2+ after removal of glutamine by dialysis, about 1 mol of radioactive iodoacetate was incorporated per mol of enzyme with inactivation. This method permits labeling of the active site for glutamine without the use of glutamine analogues.
已开发出一种两步法来标记鸡肝中甲酰甘氨酰胺核糖核苷酸（FGAR）酰胺转移酶的谷氨酰胺活性位点，该方法是先用未标记的碘乙酸酯与所有其他反应性基团反应，然后用放射性试剂对谷氨酰胺位点进行特异性标记。对该反应作为孵育时间、温度、pH以及碘乙酸酯浓度的函数进行研究后发现，在谷氨酰胺存在的情况下，该酶的两个非必需基团会发生反应，并且这种修饰后的酶对进一步的羧甲基化具有相对抗性。当通过透析去除谷氨酰胺后，将这种修饰后的酶与放射性碘乙酸酯在FGAR、ATP和Mg2+存在的情况下孵育时，每摩尔酶约有1摩尔放射性碘乙酸酯被掺入并导致失活。该方法无需使用谷氨酰胺类似物即可对谷氨酰胺的活性位点进行标记。
