Donadio S, Perks H M, Tsuchiya K, White E H
Biochemistry. 1985 May 7;24(10):2447-58. doi: 10.1021/bi00331a009.
Active-site-directed N-nitrosamides inhibit alpha-chymotrypsin through an enzyme-activated-substrate mechanism. In this work, the activation results in the release--in the active site--of benzyl carbonium ions, which alkylate and inhibit the enzyme. The final ratio of benzyl groups to enzyme molecules is 1.0, but the alkyl groups are scattered over a number of sites. Reduction and alkylation of the inhibited enzyme generate peptides insoluble in most media. Guanidine hydrochloride at 6 M proved a good solvent, and its use as an eluant on G-75 Sephadex permitted separation of the peptides. In the case of 14C-labeled enzyme, such an approach has shown that all of the alkylation occurs on the C chain of the enzyme, the chain of which the active site is constructed. Chemical modification of the peptides with ethylenediamine and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide rendered them soluble in dilute acid, permitting high-performance liquid chromatographic separation. Model studies have shown that the benzyl carbonium ions are highly reactive, alkylating amide linkages at both oxygen and nitrogen. Alkylation at oxygen produces imidate esters, which are labile centers. Hydrolysis of protein imidates results in a cleavage of the chain at that point, and separation of the peptides formed (followed by analysis) permits their identification. In our inhibition of alpha-chymotrypsin, a major site of O-alkylation has been identified as the carbonyl oxygen of Ser-214. Alkylation at the nitrogen atom of amide linkages generates stable labels; full hydrolysis with 6 N HC1 then leads to N-benzyl amino acids characteristic of those sites. Chromatography of this mixture and also 13C NMR spectroscopy of the intact inhibited enzyme have shown that three major N-alkylations have occurred. Tryptic digestion of the C chain of chymotrypsin, which contains all of the alkylation sites, provides evidence that the stable N sites are principally located between residue 216 and residue 230. These locations are consistent with predictions of alkylation sites based on inspection of a molecular model of chymotrypsin, with special reference to the aromatic binding pocket.
活性位点导向的N-亚硝基酰胺通过酶激活底物机制抑制α-糜蛋白酶。在这项工作中，激活导致苄基碳正离子在活性位点释放，这些苄基碳正离子会烷基化并抑制该酶。苄基与酶分子的最终比例为1.0，但烷基分布在多个位点上。被抑制酶的还原和烷基化产生了在大多数介质中不溶的肽。6M的盐酸胍被证明是一种良好的溶剂，将其用作G-75葡聚糖凝胶的洗脱剂可以分离这些肽。对于14C标记的酶，这种方法表明所有烷基化都发生在酶的C链上，活性位点就是由该链构建而成的。用乙二胺和N-[3-(二甲氨基)丙基]-N'-乙基碳二亚胺对肽进行化学修饰，使其可溶于稀酸，从而能够进行高效液相色谱分离。模型研究表明，苄基碳正离子具有高反应性，可烷基化氧和氮处的酰胺键。氧原子处的烷基化产生亚氨酸酯，这是不稳定的中心。蛋白质亚氨酸酯的水解导致该点处的链断裂，分离形成的肽（随后进行分析）可以对其进行鉴定。在我们对α-糜蛋白酶的抑制作用中，已确定O-烷基化的一个主要位点是Ser-214的羰基氧。酰胺键氮原子处的烷基化产生稳定的标记；用6N HCl进行完全水解，然后得到这些位点特有的N-苄基氨基酸。对该混合物进行色谱分析以及对完整的被抑制酶进行13C NMR光谱分析表明，已经发生了三种主要的N-烷基化。对包含所有烷基化位点的糜蛋白酶C链进行胰蛋白酶消化，提供了证据表明稳定的N位点主要位于残基216和残基230之间。这些位置与基于对糜蛋白酶分子模型的检查（特别参考芳香族结合口袋）对烷基化位点的预测一致。
