Hass G M, Ako H, Grahn D T, Neurath H
Biochemistry. 1976 Jan 13;15(1):93-100. doi: 10.1021/bi00646a015.
The carboxypeptidase inhibitor from Russet Burbank potatoes was subjected to a variety of chemical modifications and their effects on inhibitory activity toward carboxypeptidases A and B were determined. The importance of the alpha carboxylate of glycine-39 to the enzyme-inhibitor interaction was demonstrated by the observation that a derivative in which all four carboxyls were modified was inactive whereas a derivative in which only the beta carboxylates of aspartic acid residues 5, 16, and 17 were masked retained full inhibitory activity. In addition to these three aspartic acid residues, lysine residues 10 and 13, histidine residues 3 and 15, and arginine-32 were modified and residues 1-5 removed with little effect on inhibitory activity. Tryptophan residues 22 and 28 did not react with 2-hydroxy-5-nitrobenzyl bromide or o-nitrophenylsulfenyl chloride, and thus are presumed to be buried in the interior of the inhibitor molecule. Although tyrosine-37 was acetylated without affecting binding characteristics, both carboxypeptidases A and B protected against deacetylation by hydroxylamine. These studies indicate that the carboxyl terminal region of the inhibitor is in contact with enzyme in the complex. The parallel effects of modifications on inhibitory activity toward carboxypeptidases A and B support previous evidence that both enzymes utilize the same binding site on the inhibitor [C. A. Ryan (1971), Biochem. Biophys. Res. Commun. 44, 1265].
对来自褐皮伯班克马铃薯的羧肽酶抑制剂进行了多种化学修饰，并测定了这些修饰对其对羧肽酶A和B抑制活性的影响。通过观察发现，所有四个羧基都被修饰的衍生物无活性，而仅天冬氨酸残基5、16和17的β羧基被掩盖的衍生物仍保留完全抑制活性，这证明了甘氨酸-39的α羧基对酶-抑制剂相互作用的重要性。除了这三个天冬氨酸残基外，赖氨酸残基10和13、组氨酸残基3和15以及精氨酸-32也被修饰，并且去除了残基1-5，对抑制活性影响不大。色氨酸残基22和28不与2-羟基-5-硝基苄基溴或邻硝基苯硫酰氯反应，因此推测它们埋在抑制剂分子内部。尽管酪氨酸-37被乙酰化而不影响结合特性，但羧肽酶A和B都能防止其被羟胺脱乙酰化。这些研究表明，抑制剂的羧基末端区域在复合物中与酶接触。修饰对羧肽酶A和B抑制活性的平行影响支持了先前的证据，即这两种酶在抑制剂上利用相同的结合位点[C. A. Ryan（1971年），《生物化学与生物物理研究通讯》44，1265]。
