Enzyme intermediates reactivity

Figure 8 Irreversible inhibitors of proteases. Serine and cysteine proteases can be acylated by aza-peptides, which release an alcohol, but cannot be deacylated due to the relative unreactivity of the (thio) acyl-enzyme intermediate. Reactive carbons, such as the epoxide of E64, can alkylate the thiol of cysteine proteases. Phosphonate inhibitors form covalent bonds with the active site serine of serine proteases. Phosphonates are specific for serine proteases as a result of the rigid and well-defined oxyanion hole of the protease, which can stabilize the resulting negative charge. Mechanism-based inhibitors make two covalent bonds with their target protease. The cephalosporin above inhibits elastase [23]. After an initial acylation event that opens the p-lactam ring, there are a number of isomerization steps that eventually lead to a Michael addition to His57. Therefore, even if the serine is deacylated, the enzyme is completely inactive.

In the chymotrypsiii mechanism, the nitrophenylacetate combines with the enzyme to form an ES complex. This is followed by a rapid second step in which an acyl-enzyme intermediate is formed, with the acetyl group covalently bound to the very reactive Ser . The nitrophenyl moiety is released as nitrophenolate (Figure 16.22), accounting for the burst of nitrophenolate product. Attack of a water molecule on the acyl-enzyme intermediate yields acetate as the second product in a subsequent, slower step. The enzyme is now free to bind another molecule of nitrophenylacetate, and the nitrophenolate product produced at this point corresponds to the slower, steady-state formation of product in the upper right portion of Figure 16.21. In this mechanism, the release of acetate is the rate-llmitmg step, and accounts for the observation of burst kinetics—the pattern shown in Figure 16.21. 

Aromatase inhibitors may be classified into two types. Type 1 aromatase inhibitors bind to the aromatase enzyme irreversibly, so they are called inactivators. In some cases they are dubbed mechanism-based or suicide inhibitors when they are metabolized by the enzyme into reactive intermediates that bind covalently to the active site. Type 1 aromatase inhibitors are usually steroidal in structure as represented by exemestane (1), formestane (13), and atamestane (14). Formestane (13) was launched by Ciba-Geigy in 1992. As formestane (13) is readily and extensively metabohzed when administered orally, it is used as a depot formulation for injection. 

The first suggestion of a practical form of antidotal therapy came in 1949 from Hestrin, who found that acetylcholinesterase (AChE) catalyzed the formation of acetohydroxamlc acid when incubated with sodium acetate and hydroxylamine. Critical in vitro studies in the next decade led to the development of a practical approach to therapy. The crucial concept in these studies was the recognition that the compound formed when AChE reacted with a phosphorus ester was a covalent phosphoryl-enzyme Intermediate similar to that formed in the hydrolysis of acetylcholine. 3 Wilson and colleagues, beginning in 1951, demonstrated that AChE inhibited by alkyl phosphate esters (tetraethyl pyrophosphate, TEPP) could be reactivated by water, but that free enzyme formed much more rapidly in the presence of hydroxylamine. 0 21 Similar results... 

In addition to participating in acid-base catalysis, some amino acid side chains may enter into covalent bond formation with substrate molecules, a phenomenon that is often referred to as covalent catalysis.174 When basic groups participate this may be called nucleophilic catalysis. Covalent catalysis occurs frequently with enzymes catalyzing nucleophilic displacement reactions and examples will be considered in Chapter 12. They include the formation of an acyl-enzyme intermediate by chymotrypsin (Fig. 12-11). Several of the coenzymes discussed in Chapters 14 and 15 also participate in covalent catalysis. These coenzymes combine with substrates to form reactive intermediate compounds whose structures allow them to be converted rapidly to the final products. 

Inhibition of the type I deiodinase by PTU is uncompetitive with substrate and competitive with cofactor. This is the case for the ORD of T4 and rT3 as well as for the IRD of T3 and T3S [7,8]. Persistent inactivation of enzyme by PTU and covalent labelling with radioactive inhibitor requires the presence of substrate and is only reversed with high DTT [42,47], All available evidence indicates that PTU reacts with a substrate-induced enzyme intermediate. As thiourea derivatives are particularly reactive towards sulfenyl iodide (SI) groups, generation of an enzyme-SI intermediate is thought to precede thiouracil inhibition through mixed disulfide formation [7,8]. 

Efficient modification steps through the proper orientation of the inhibitor reactive group to the enzyme nucleophile is realized by covalent bond formation. A classic example of this type is the modification of a methionine residue of chymotrypsin by /7-nitrophenyl bromoacetyl a-aminoisobutyrate (26)47). In this instance, the reactive group (bromoacetyl) is fixed at the locus near the active site through a covalent bond by means of acyl enzyme intermediates. 

Figure 5 Action of p-lactam antibiotics. Cell wall biosynthetic transpeptidases activate peptidoglycan peptides for cross-linking by formation of a covalent enzyme intermediate (A). The reactivity of the active-site Ser nucleophile is exploited by p-lactam antibiotics such as penicillin G (shown) to form a hydrolytically stable acylenzyme (B).

One remarkable feature of enzyme-catalyzed reactions is the ability to generate at enzyme sites reactive intermediates, such as carbanions, carbocations, and radicals, that normally would require strong reaction conditions to generate in chemical reactions and would be very unstable in aqueous solution. 

A more narrowly defined group within this second set of compounds is the set of mechanism-activated inhibitors. These are compounds which do not have a second pre-existing reactive functionality. Rather, they use the normal catalytic machinery of the enzyme to generate, or unmask, a reactive species in the acyl-enzyme intermediate (E I). This new species then alkylates a second, suitably placed, active-site residue and permanently inactivates (binds to) the enzyme (even if deacylation of Ser-195 subsequently occurs). Efficient mechanism-activated inhibitors are those which have a high ratio of alkylation (Atj) to release k of the active enzyme. Because the second reactive functionality is only generated in the active site,... 

Chloramphenicol can interfere with the ehmination of drugs that are inactivated by hepatic metabolism, probably through a mechanism involving inhibition of microsomal enzymes. The mechanism has been claimed to be inactivation of microsomal enzymes via an intermediate reactive metabolite that binds covalently to the protein moiety of cjdochrome P450 (71). Assuming such a mechanism, chloramphenicol would be expected to interact with the metabolism of other drugs dealt with by cytochrome P450. 

As one may expect from the peroxidase reaction mechanism described in Eqs. (2-4), the reactivity of the enzyme intermediates towards a particular substrate may be estimated a priori on the basis of the thermodynamic driving force of these two electron-transfer reactions, which is directly related with the difference between the oxidation/reduction potentials of both the enzyme active intermediates (i.e.. Col and Coll) and the substrate radicals. Thus, the thermodynamic driving force for the reaction of Col (or Coll) with the reducing substrates is the difference between the mid-point potentials of the CoI/CoII (or CoII/Felll) and the substrate radical/substrate (R, Hr/RH) redox couples ... 

Kinetically controlled syntheses, which are more often studied, can only be carried out by enzymes forming a reactive acyl-enzyme intermediate (serine or cysteine protease. Scheme 5). The reaction starts with weakly activated amino acids (e.g. esters), and the rapidly formed reactive intermediate RCOE is attacked by nucleophiles like amines and water. If k-i and A 4[H2NR ] > < 3[H20], the desired peptide accumulates. Short reaction times, low enzyme concentrations and the danger of secondary hydrolysis of the peptide product are characteristics of these reactions. The optimal pH usually lies above pH 8. 

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