Chymotrypsin inhibitors affinity labels

More specific evidence came from affinity labeling with molecules which could react with specific amino acid group sat or adjacent to the substrate site. These labels were substrate analogues and competitive inhibitors. Substituted aryl alkyl ketones were used. TV-p-toluene-sulphonyl-L-phenylalanine chloromethyl ketone (TPCK) blocked the activity of chymotrypsin. Subsequent sequence analysis identified histidine 57 as its site of binding (see Hess, 1971, p 213, The Enzymes, 3rd ed.). Trypsin, with its preference for basic rather than aromatic residues adjacent to the peptide bond, was not blocked by TPCK but was susceptible to iV-p-toluenesulphonyl-L-lysine chloromethyl ketone (TLCK) (Keil, ibid, p249). 

The most interesting class of protease inhibitors in viral studies has been the chloromethyl ketone derivatives of amino acids (54j 55) These were designed as affinity labels of serine-type proteases, and react irreversibly with histidine and serine residues in the active sites of proteases. There is a basis for selectivity of the chloromethyl ketones phenylalanyl and lysyl derivatives were synthesized, which had specificity for chymotrypsin and trypsin, respectively (54) This specificity led to studies on inhibition of poliovirus protein cleavage, with positive results (25, 26). 

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