Chloromethyl ketone substrate

J. Drenth, K. H. Kalk, and H. M. Swen. Binding of chloromethyl ketone substrate analogues to crystalline papain. Biochemistry 75 3731 (1976). 

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 chloromethyl ketone-based inhibitor-complex crystal structures suggested the strand E993-P996 as a recognition strand for the unprimed-substrate residues. 

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. 

Fig. 11.10 (A) Burst kinetics for release of the leaving group from a substrate of a-chymotrypsin. (B) Titration of papain with the irreversible inhibitor 4-toluenesulphonamidomethyl chloromethyl ketone using methyl benzoylglycinate as substrate.

An experiment with an irreversible inhibitor should carry with it a control experiment involving the addition of a substrate if the location of the reaction with inhibitor is at the active site, then the addition of a substrate will slow down the rate of inhibition. For example, the reactivity of papain (5 pM) with a 1.71 pM solution of 4-toluenesulphonylamidomethyl chloromethyl ketone suffers a drop of 1.68-fold when the substrate (methyl hippurate) is changed from 12.7 to 21.1 mM. The inhibitor which reacts covalently with the enzyme should carry either a radioactive or spectroscopic tag which would enable the location of the altered amino acid to be determined in the sequence, and hence in the three-dimensional X-ray crystallographic map of the enzyme. An alternative approach is to design an inhibitor with groups (analogous to those attached to the substrate) which force it to bind at the active site (Scheme 11.18). 

Note Reactions were performed under the conditions described in the text with either colorimetric peptide pNA4 (250 pM) or fluorogenic peptide F3 (35 pM) as a substrate. The IC50 values represent the inhibitor concentration required to reduce the protease activity by 50% of the control containing no inhibitor. NI, no inhibition was observed at the concentrations indicated. E64, frans-epoxysuccinyl-L-leucylamide-(4-guanidino)-butane PMSF, phenylmethylsulphonyl fluoride TLCK, tosyl-L-lysine-chloromethyl ketone. 

Peptide Chloromethyl Ketones. Peptide chloromethyl ketone inhibitors have been studied extensively and a fairly detailed picture of the inhibition reaction (see Figure 3) has emerged from numerous chemical and crystallographic studies (30,31). The inhibitor resembles a serine protease substrate with the exception that the scissile peptide bond of the substrate is replaced with a chloromethyl ketone functional group in the inhibitor. The inhibitor binds to the serine protease in the extended substrate binding site and the reactive chloromethyl ketone functional group is placed then in the proper position to alkylate the active-site histidine residue. In addition, the serine OH reacts with the inhibitor carbonyl group to form a hemiketal. 

Peptide chloromethyl ketone inhibitors have been developed for almost every serine protease that has been characterized adequately (30). For example, human leukocyte elastase, due to its involvement in emphysema, has been studied extensively with this class of inhibitor (32). The rate at which peptide chloromethyl ketones inhibit elastase is influenced by their interaction with the primary substrate binding site (Si) of the enzyme and by interactions at other subsites. The most effective chloromethyl ketone elastase inhibitor found thus far is MeO-Suc-Ala-Ala-Pro-ValCH2Cl (MeO-Suc- = CH3OCOCH2CH2CO-). This will not inhibit the other major leukocyte protease, cathepsin G (see Table VI). In contrast, Z-Gly-Leu-Phe-CH2C1 (Z = C6H5CH2OCO-) inhibits cathepsin G, but not elastase. Both enzymes can be inhibited with Ac-Ala-Ala-Pr o-V alCH2Cl. 

Figure 3. Reaction of a serine protease with a peptide chloromethyl ketone. The side chain of the Pt residue of the inhibitor is shown interacting with the primary substrate binding subsite (SJ of the enzyme.

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.

A very convenient hydroxymethylation process has been developed based on the Sml2-mediated Bar-bier-type reaction. Treatment of aldehydes or ketones with benzyl chloromethyl ether in the presence of Smh provides the alkoxymethylated products in good to excellent yields. Subsequent reductive cleavage of the benzyl ether provides hydroxymethylated products. Even ketones with a high propensity for enolization can be alkylated by this process in reasonable yields. The method was utilized by White and Soners as a key step in the synthesis of ( )-deoxystemodinone (equation 27). This particular ketone substrate resisted attack by many other nucleophilic reagents (such as methyllithium) owing to conpeti-tive enolate formation. 

K., Neurath, H. and Woodbury, RG. (1985). Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dc skin proteases and human cathepsin G with peptide-4-nitroanilide substrates and with peptide chloromethyl ketone and sulphonyl fluoride inhibitors. Biochemistry 24, 2048-2058. 

Cinchonidine (99) has extended the substrate scope of the ketone conjugate additions to P-substituted methylidene malononitriles. In particular, the reaction of a-chloromethyl ketones, under very low loading conditions, affords tetrasubstituted cyclopropanes in moderate to good enantioselectivities after intramolecular cycliza-tion (Scheme 2.45) [142], A similar strategy has been followed to synthesize, with moderate to good enantioselectivities (56-90% ee) optically active naphlhopyran derivatives by a conjugate addition/cyclization sequence between 2-naphthol and a,a-dicyanoolefins [ 143 ]. 

Studies of y-chymotrypsin by Segal et al and of subtilisin by Kraut et al. with chloromethyl ketone analogues of good phenylalanine polypeptide substrates have indicated further similarities in substrate binding and specificity of these enzymes. It has previously been reported that both enzymes contain a serine at the active site which is acylated by ester substrates, a histidine hydrogen-bonded to this serine which is alkylated by active-site directed halogenomethyl ketones, and an aspartate which is buried and hydrogen-bonded not only to the active-site histidine but also in each case to a further serine. 

Enzymes cleaving polypeptide substrates at the carboxyl group of arginine residues can be irreversibly inhibited by two arginine chloromethyl ketones, P-NO2-ZACK and TACK. The reaction is specifically oriented to their active sites. 

Luciferase, 151, 287, 537-541 Luciferin, ethylation of, 537-541 Lysine chloromethyl ketone, 609, 611, 612 Lysozyme, 403-414 affinity labeling of, 410-413 binding site, 75, 411, 412 physical properties, 404 substrate specificity, 404 Lysylalanyllysylchloromethyl ketone, 206 Lysylchloromethyl ketone, 201... 

Bums and Turner (2) subjected proteins to either alkylation with iodo-acetic acid (3) or performic acid oxidation (4) to render them susceptible to enzymatic digestion. The treated proteins were then dissolved in ammonium bicarbonate buffer (0.05 M, pH 8.4) to a concentration of 2 mg/ml and TPCK-treated trypsin [L-(l-tosylamide-2-phenylethyl chloromethyl ketone] was added to give a final enzyme-to-substrate ratio of 1 75. The digest is incubated for 5 h at 30 C, freeze-dried, and redissolved in 10% isopropanol for application to the plates. 

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