Chymotrypsin affinity labeling

Figure 7. Two examples of irreversible inactivators that are not suicide substrates a) TPCK, a classic" affinity label of the serine protease chymotrypsin, b) ZFK-CH2-mesitoate, a quiescent" affinity label of the cysteine protease cathepsin B, and c) the kinetic scheme for both forms of affinity label-inactivation.

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 principles of the method are very nicely illustrated by one of the first affinity labeling experiments, the reaction of /exs-i.-phenylalanine chloro-methyl ketone (TPCK) with chymotrypsin.1 TPCK resembles substrates like fexs-L-pheny 1 alanine methyl ester, but the chloromethyl ketone group of TPCK is an alkylating reagent. 

Halomethyl ketones and acids are known to react with thiols and imidazoles. TPCK reacts far more rapidly with chymotrypsin than it does with normal histidine-containing peptides because of its high reactivity as an affinity label. This can be seen in Table 9.2 for an analogous chloromethyl ketone. In addition to this important diagnostic feature, the irreversible inhibition of chymotrypsin by TPCK has four other characteristic features 1,4... 

X less reactive than—S". Useful in affinity labels cf. chymotrypsin, p. 278. 

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They are thus more specific for the enzyme active site than are group-specific reagents. Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analog for chymotrypsin (Figure 8.21). TPCK binds at the active site and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme. The compound 3-bromoacetol is an affinity label for the enzyme triose phosphate isomerase (TIM). It mimics the normal substrate, dihydroxyacetone phosphate, by binding at the active site then it covalently modifies the enzyme such that the enzyme is irreversibly inhibited (Figure 8.22). 

Figure 8.21. Affinity Labeling. (A) Tosy 1-1-phenylalanine chloromethyl ketone (TPCK) is a reactive analog of the normal substrate for the enzyme chymotrypsin. (B) TPCK binds at the active site of chymotrypsin and modifies an essential histidine residue.

The striking successes achieved by Shaw (1970a) and his coworkers with haloketone derivatives of N-tosyl-phenylalanine and a-N-tosyl-lysine as affinity labels for chymotrypsin and trypsin, respectively, have stimulated their use in a large number of affinity labels. Haloketones are potentially reactive with all the nucleophilic amino acid residues in proteins. Examples of residues modified by haloketones include methionine (Sigman et al. 1970), glutamate (Visser et al. 1971), cysteine (Porter et al. 1971), histidine (Schoellman and Shaw 1963) and serine (Schroeder and Shaw 1971). 

Since epoxides potentially will react with a variety of amino acids, many products may be formed if they are incorporated into affinity labels. However at present affinity labels containing epoxides have been shown to modify either glutamate or aspartate residues in pepsin, lysozyme, those phosphate isomerase and j -glucosidase. The only other residue which has as yet been modified by epoxides is methionine 192 of chymotrypsin. In general, the other possible products should be similar in stability to derivatives formed by haloketones. Therefore the methods for identification of the amino acid derivatives formed by reaction with epoxides closely parallel those described in connection with haloketones ( 5.3.2). 

In contrast, 5-benzyl-6-chloro-2-pyrone is not a substrate for chymotrypsin, but inactivates the enzyme with the formation of a shoulder in the absorbance spectrum at 320 nm (Westkaemper and Abeles, 1983). The chromophore, which is similar to that of the pyrone ring, appears with kinetics corresponding to the rate of inactivation and is lost on reactivation of the enzyme. The crystal structure and C NMR studies of the modified enzyme demonstrate that the benzyl group is bound in the specificity pocket of the active site and that serine-195 is covalently attached to C-6 of the intact pyrone (Ringe et ai, 1985) (Fig. 49). Inactivation by the 5-benzyI analog is therefore an example of affinity labeling rather than mechanism-based inactivation and emphasizes the importance of the side chain of protease inactivators in proper orientation of the compound in the active site. 

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). 

Figure 1, Panel A shows the RP-HPLC chromatogram of the combined digest (Endo Lys-C followed by chymotrypsin) of MIANS-ricin A-chain detected at 214 and 320 nm. There are only 6 major peptides that are MIANS-labeled as evidenced by the 320 nm profile. All of these peptides bind to and are eluted from the anti-MlANS column as shown in Panel B of Figure 1. Moreover, as the profile at 214 nm shows, there is no evidence of significant amounts of additional, unlabeled peptides sticking to the affinity column. Note also that the ratio of the peaks in the 320 nm chromatogram of Panel B is the seune as that in the 320 nm chromatogram of Panel A, indicating that each of the MIANS-labeled peptides binds to the affinity column equivalently.

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