Peterson R G, Richards F F, Handschumacher R E
J Biol Chem. 1977 Mar 25;252(6):2072-6.
The L-asparagine analogue 5-diazo-4-oxo-L-[5-14C]norvaline binds irreversibly to the active site of Escherichia coli L-asparaginase. Conditions for optimal labeling in buffers containing 50% dimethylsulfoxide have been developed and kinetic parameters of the inactivation have been determined. After reduction, alkylation and subsequent degradation of the modified enzyme with alpha-chymotrypsin, the principal radioactive decapeptide of sequence Val-Gly-Ala-Met-Arg-Pro-Ser-Thr-Ser-Met was isolated. A second radioactive hexapeptide Arg-Pro-Ser-Thr-Ser-Met resulting from chymotryptic digestion of the decapeptide was also isolated. Evidence is presented for the attachment of the 5-diazo-4-oxo-L-norvaline residue to serine-9 in the decapeptide via an acid-labile linkage.
L-天冬酰胺类似物5-重氮-4-氧代-L-[5-¹⁴C]正缬氨酸与大肠杆菌L-天冬酰胺酶的活性位点不可逆结合。已开发出在含50%二甲基亚砜的缓冲液中进行最佳标记的条件，并测定了失活的动力学参数。在用α-胰凝乳蛋白酶对修饰后的酶进行还原、烷基化及随后的降解后，分离出了序列为Val-Gly-Ala-Met-Arg-Pro-Ser-Thr-Ser-Met的主要放射性十肽。还分离出了由该十肽经胰凝乳蛋白酶消化产生的第二种放射性六肽Arg-Pro-Ser-Thr-Ser-Met。有证据表明5-重氮-4-氧代-L-正缬氨酸残基通过酸不稳定键连接到十肽中的丝氨酸-9上。
