Alkyl-enzyme intermediate

H. F. Tzeng, L. T. Laughlin, S. Lin, R. N. Armstrong, The Catalytic Mechanism of Microsomal Epoxide Hydrolase Involves Reversible Formation and Rate-Limiting Hydrolysis of the Alkyl-Enzyme Intermediate , J. Am. Chem. Soc. 1996, 118, 9436 -9437. 

Tzeng H-F, Laughlin LT, Lin S, Armstrong RN. The catalytic mechanism of microsomal epoxide hydrolase involves reversible formation and rate-limiting hydrolysis of the alkyl-enzyme intermediate. J Am Chem Soc 1996 118 9436-9437. 

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... 

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.

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,... 

The first step in peroxidase catalysis is the formation of a two-electron oxidized enzyme intermediate on reaction of peroxide (ROOH, R = H, alkyl, or aryl) with the resting ferric form of the enzyme. This intermediate, which stores the two oxidizing equivalents of the peroxide, is termed compound I. An Fe -OOH intermediate, or ES complex. 

One biological reaction that involves radicals is the one responsible for the conversion of toxic hydrocarbons to less toxic alcohols. Carried out in the liver, the hydroxyl-ation of the hydrocarbon is catalyzed by an iron-porphyrin-containing enzyme called cytochrome P450 (Section 12.8). An alkyl radical intermediate is created when Fe O abstracts a hydrogen atom from an alkane. In the next step, Fe OH dissociates homolytically into Fe and HO, and the HO immediately combines with the radical intermediate to form the alcohol. 

A phosphoryl-enzyme intermediate is formed during reactions catalysed by alkaline phosphatase. 0-4-Nitrophenyl phosphorothioate is hydrolysed 1000 times more slowly by alkaline phosphatase than is its oxygen analogue, suggesting an 5n2(P) mechanism for the phosphorylation of the enzyme. From a kinetic study of the reaction between a series of alkyl-, aryl-, and arylamido-phosphates and alkaline phosphatase, it has been shown that steric factors are important in these reactions. Moreover, although amidophosphates [e.g. (69)] are substrates for this enzyme ... 

Concerning the reactions of L-lactate monomers, alkyl L-lactates were not consumed at aU in the oligomerization. In the hydrolysis, alkyl L-lactates were hydrolyzed to give L-lactic acid [step (h) in Fig. 3]. This is a clear indication that step (e) actually took place to produce [Sj-acyl-enzyme intermediate EM. However, neither the OH group of D-lactate nor the OH group of L-lactate was allowed to attack EM to give L,D-dimer via step (f) or Lj.-dimer via step (g). 

Figure 3.2 Schematic representation of the reactions between sarin and AChE. (a) Sarin and the active site of AChE combine to form an inhibitor-enzyme intermediate, (b) The fluoride has been lost, leaving a complex of sarin and AChE. From this state, either (c) spontaneous hydrolysis and restoration of function or (d) dealkylation can occur, (c) The ester link in the phosphonylated AChE has been hydrolysed, the enzyme has reactivated and alkyl methyl phosphonic acid has been formed, (d) The link between the alkyl group and the phosphorus has been cleaved. This produces a conformational change that results in the formation of a very stable agent-enzyme complex that is then resistant to spontaneous hydrolysis and reactivation by oximes. This is known as ageing . The rate of ageing is dependent on the nature of the alkyl group and is fairly slow (hours) in the case of sarin and VX, but is very rapid (minutes) in the case of soman. Figure adapted from Vale et aL ...

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