Inglese J, Smith J M, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16801.
Biochemistry. 1990 Jul 17;29(28):6678-87. doi: 10.1021/bi00480a018.
The affinity reagent N10-(bromoacetyl)-5,8-dideazafolate has previously been shown to inactivate glycinamide ribonucleotide transformylase (EC 2.1.2.2) from Escherichia coli in an active-site-directed manner with a 1:1 stoichiometry [Inglese et al. (1990) Biochemistry 29, 1436-1443]. After a series of mild proteolytic digestions, the dideazafolate label was localized to an active-site peptide attached by an ester linkage to the highly conserved residue Asp 144. Subsequent site-specific mutagenesis of Asp 144 to Asn 144 resulted in a catalytically inactive enzyme that retained the ability to bind substrates and inhibitors. The Asn 144 mutant could be further labeled with the affinity reagent in an active-site-directed stoichiometric fashion; however, the site of modification in this case was His 119. These results imply that Asp 144 may function as a general base within the catalytic center of the transformylase and is in close proximity to His 119 in the folded protein.
亲和试剂N10-(溴乙酰基)-5,8-二氮杂叶酸先前已被证明能以活性位点导向的方式使来自大肠杆菌的甘氨酰胺核糖核苷酸转甲酰基酶（EC 2.1.2.2）失活，化学计量比为1:1 [英格利斯等人（1990年），《生物化学》29卷，1436 - 1443页]。经过一系列温和的蛋白酶消化后，二氮杂叶酸标记定位到一个通过酯键与高度保守的天冬氨酸144相连的活性位点肽段上。随后将天冬氨酸144定点突变为天冬酰胺144，得到一种催化无活性的酶，该酶仍保留结合底物和抑制剂的能力。天冬酰胺144突变体可以以活性位点导向的化学计量方式进一步用亲和试剂标记；然而，在这种情况下修饰位点是组氨酸119。这些结果表明，天冬氨酸144可能在转甲酰基酶的催化中心起通用碱基的作用，并且在折叠的蛋白质中与组氨酸119紧密相邻。
