Alkylation of amide linkages and cleavage of the C chain in the enzyme-activated-substrate inhibition of .alpha.-chymotrypsin with N-nitrosamides

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Abstract
Active-site-directed N-nitrosamides inhibit .alpha.-chymotrypsin through an enzyme-activated-substrate mechanism. 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 showed 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 showed 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 the inhibition of .alpha.-chymotrypsin, a major site of O-alkylation was identified as the carbonyl oxygen of Ser-214. Alkylation at the nitrogen atom of amide linkages generates stable labels; full hydrolysis with 6N HCl 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 showed that 3 major N-alkylations 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.