Titani K, Koide A, Ericsson L H, Kumar S, Hermann J, Wade R D, Walsh K A, Neurath H, Fischer E H
Biochemistry. 1978 Dec 26;17(26):5680-93. doi: 10.1021/bi00619a014.
This communication presents the strategy and experimental details which establish the amino acid sequence of the carboxyl-terminal 492 residues (residues 350 through 841) of rabbit muscle glycogen phosphorylase (EC 2.4.1.1). The heavy segment (Hs), derived from the native enzyme by limited proteolysis with subtilisin, was cleaved with cyanogen bromide to yield 15 fragments. The amino acid sequences of 12 of these are described herein. The sequence of 3 other fragments (CB17C, CB18, and CB15) is described in accompanying reports by Koide, A., et al., and Hermann, j., et al. ((1978) Biochemistry 17 (first and second papers, respectively, in a series in this issue)). These 15 fragments were aligned by analysis of three others generated by cleavage of the heavy segment Hs at asparaginylglycine bonds with hydroxylamine and of four more generated by acid cleavage of aspartylproline bonds. Lysine-679 was identified as the binding site of the essential cofactor pyridoxal 5'-phosphate. These data, together with those reported in the accompanying papers (vide supra), establish the complete sequence of the 841 amino acid residues in glycogen phosphorylase. They provide a chemical basis on which the relationship between structure and function of the enzyme can be examined.
本通讯介绍了确定兔肌肉糖原磷酸化酶（EC 2.4.1.1）羧基末端492个残基（第350至841位残基）氨基酸序列的策略和实验细节。通过枯草杆菌蛋白酶有限水解从天然酶衍生而来的重链片段（Hs），用溴化氰裂解产生15个片段。本文描述了其中12个片段的氨基酸序列。另外3个片段（CB17C、CB18和CB15）的序列在小出、A.等人和赫尔曼、J.等人的相关报告中描述（（1978年）《生物化学》17卷（分别为本期刊系列中的第一篇和第二篇论文））。通过分析重链片段Hs在天冬酰胺基甘氨酸键处用羟胺裂解产生的另外3个片段以及在天冬氨酸基脯氨酸键处酸裂解产生的另外4个片段，将这15个片段进行了比对。赖氨酸-679被确定为必需辅因子磷酸吡哆醛的结合位点。这些数据与相关论文（见上文）中报道的数据一起，确定了糖原磷酸化酶中841个氨基酸残基的完整序列。它们为研究该酶的结构与功能之间的关系提供了化学基础。
