Inhibition peptide chloromethyl ketone

Powers, J. C., et al. 1977. Specificity of porcine pancreatic elastase, human leukocyte elastase and cathepsin G. Inhibition with peptide chloromethyl ketones. Biochim Biophys Acta 485 156. 

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. 

Peptide Fluoromethyl Ketones Fluoroalkyl derivatives of the peptide chloromethyl ketones have been prepared in an attempt to improve specificity by reducing nonspecific alkylation at cysteine residues (Rasnick, D., Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B, Anal. Biochem. 149, 461 65, 1985). Nonspecific reaction with sulfydryl groups such as those in glutathione was reduced there was still reaction with active site cysteine although at a slower rate than with the chloroalkyl derivative (16,200 M s vs. 45,300 1 2 21.9 min. vs. 

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... 

The diazomethyl ketone functional group was first observed to be an affinity label by Buchanan and co-workers who showed that the antibiotic azaserine, an O-diazoacetyl derivative, 9 inhibited an enzyme in the biosynthesis of purine by alkylation of a cysteine residue. 10 The acid protease pepsin was then observed to be inhibited by peptidyl diazomethyl ketones in the presence of copper ions with the resulting esterification of an aspartate residue. 11 Two peptidyl diazomethyl ketones, Z-Phe-CHN2 and Z-Phe-Phe-CHN2, were found to irreversibly inactivate papain, a cysteine protease. 12 Since these reports, many peptidyl diazomethyl ketones have been prepared primarily as inhibitors of various cysteine proteases. 7 Peptidyl diazomethyl ketones are also synthetic intermediates and have been used to prepare chloromethyl ketones (Section 15.1.3), 13 bromomethyl ketones (Section 15.1.3), acyloxymethyl ketones, 14 and (i-peptides. 15 A few peptidyl diazoalkyl ketones have been reported. 16,17 ... 

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. 

In the past ten years, there has been developed a series of enzyme inhibitors that combine the features of an alkylating agent with specificity for the active site of an enzyme, thus permitting alkylation and identification of a group at or near the active center of an enzyme, or a particular enzyme to be specifically inactivated. Thus a l-chloro-4-phenyl-3-p-toluenesulfonamido-2-butanone ( W-p-tolylsulfonylphenylalanine chloro-methyl ketone ) inactivates chymotrypsin (which cleaves a peptide bond adjacent to an aromatic residue), and 7-amino-l-chloro-3-p-toluene-sulfonamido-2-heptanone ( a-iV-p-tolylsulfonyllysine chloromethyl ketone ) inhibits trypsin (which cleaves a peptide bond adjacent to lysine. In both cases, a histidine residue at the active site is alkylated, and neither inhibitor will inhibit the other enzyme at low concentrations. 

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