Mariano P S, Glover G I, Petersen J R
Biochem J. 1978 Apr 1;171(1):115-22. doi: 10.1042/bj1710115.
A reinvestigation of the modification reactions of alpha-chymotrypsin with phenacyl bromide was carried out. Results conclusively demonstrate that the chemically and physically different modified enzymes prepared at pH 4 and at pH 7 both contain the phenacyl group at methionine-192 in the sulphonium salt form. Evidence to suppoort this conclusion derives from 13C nuclear-magnetic-resonance spectroscopic observations on [methylene-13C]phenacyl-enriched enzymes. More conclusively, the methionine-192-containing C-chain, derived by performic acid oxidative cleavage of radioactively-labelled enzyme prepared at pH 7, was shown to contain the phenacyl moiety and to undergo dealkylation by 2-mercaptoethanol with loss of this moiety. In addition, thermolytic cleavage of the high-pH enzyme results in fragmentation of the polypeptide chain in a fashion analogous to model reactions of phenacylmethionyl dipeptides and other methionine-192 sulphonium salts. A rationalization of the unusual nature of the high-pH phenacyl-modified enzyme based on the irreversible formation of stable conformation in which the phenacyl moiety is rigidly located in interior regions of the enzyme is presented and discussed.
对α-胰凝乳蛋白酶与苯甲酰溴的修饰反应进行了重新研究。结果确凿地表明，在pH 4和pH 7条件下制备的化学和物理性质不同的修饰酶在甲硫氨酸-192处均以鎓盐形式含有苯甲酰基。支持这一结论的证据来自对富含[亚甲基-¹³C]苯甲酰基的酶的¹³C核磁共振光谱观察。更确凿的是，通过对在pH 7条件下制备的放射性标记酶进行过甲酸氧化裂解得到的含甲硫氨酸-192的C链，显示含有苯甲酰基部分，并会被2-巯基乙醇脱烷基化而失去该部分。此外，高pH值酶的热解裂解导致多肽链断裂，其方式类似于苯甲酰甲硫氨酰二肽和其他甲硫氨酸-192鎓盐的模型反应。基于苯甲酰基部分刚性地位于酶内部区域的稳定构象的不可逆形成，对高pH值苯甲酰基修饰酶的异常性质进行了合理化解释并进行了讨论。
