Pseudoirreversible enzyme inhibitors

A significant difference between pseudoirreversible inhibitors and mechanism-based inactivators is the reversibiUty of the inactivation. A complete evaluation of the mechanism involved would require evidence not only for the covalent enzyme-inhibitor complex, but also for its decomposition products and its rate of reactivation. It is often difficult to identify the active site amino acid residue covalently linked to the inhibitor because of the instabiUty of the complex. 

Pseudoirreversible inhibitors are the least common of the covalently binding enzyme inhibitors. They have some features in common with both affinity labels (Section 3.2) and mechanism-based inhibitors (Section3.3) but they have one distinguishing feature that is. 

As may be expected, criteria for the study of pseudo irreversible inhibitors are very similar to those for both affinity labels and mechanism-based inhibitors. However, because of the inherent reversibility of pseudoirrevers-ible inhibitors, it may be more difficult to obtain structural evidence for the covalent enzyme inhibitor adduct. Further, determination of the rate of reactivation and characterization of the products of the recovery process will also be of major importance in designating an inhibitor as pseudoirreversible. 

Inhibition of cathepsin H by the inhibitor is rapid, reaching maximal levels within 15 seconds after addition of the inhibitor at 0°C. Kinetical studies indicate that the purified inhibitor inhibited papain noncom-petitively and pseudoirreversibly. The inhibitor inhibits cathepsin H by forming an enzyme- inhibitor complex in a molar ratio of 1 1. When cathepsin H is preincubated with E-64, formation of the enzyme-inhibitor complex is retarded. Conversely, when H-labeled E-64 is added to the reaction mixture after the enzyme-inhibitor complex is formed, incorporation of H-labeled E-64 into cathepsin H is slight. Those results suggest that the thiol proteinase inhibitor binds to the active site region of thiol proteinases. 

An example of a pseudoirreversible inhibitor has been demonstrated for chymotrypsin (36). This enzyme is a serine protease, and its mechanism of catalysis may be outlined as follows, where or R2 preferentially is a hydrophobic amino acid residue. 

In pseudoirreversible inhibitors of the second class, the enzyme is regenerated by the inhibitor simply dissociating from the enzyme that is, he binding is covalent but reversible (A 2 k,). This class can also be ex-... 

The affinity of the inhibitor for AcChE could be decreased (with a concomitant increase in the value of k,) by sequentially reducing the number of fluorine atoms into the methyl group adjacent to the ketone (220). Finally, it should be noted that the two classes of pseudoirreversible inhibitor can be differentiated by examiningthe decomposition products of the inhibition reaction. When hydrolysis is required for enzyme regeneration, cleavage products, such as substituted carbamates, will be in evidence. Conversely, the trifluoromethyl ketones will not be broken down by AcChE and no decomposition products will be observed. 

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