Epoxide hydrolases reaction mechanisms

A critical input in unraveling the catalytic mechanism of epoxide hydrolases has come from the identification of essential residues by a variety of techniques such as analysis of amino acid sequence relationships with other hydrolases, functional studies of site-directed mutated enzymes, and X-ray protein crystallography (e.g., [48][53][68 - 74]). As schematized in Fig. 10.6, the reaction mechanism of microsomal EH and cytosolic EH involves a catalytic triad consisting of a nucleophile, a general base, and a charge relay acid, in close analogy to many other hydrolases (see Chapt. 3). 

L. T. Laughlin, H. F. Tzeng, S. Lin, R. N. Armstrong, Mechanism of Microsomal Epoxide Hydrolase. Semifunctional Site-Specific Mutants Affecting the Alkylation Half-Reaction , Biochemistry 1998, 37, 2897 - 2904. 

The use of enzymes and whole cells as catalysts in organic chemistry is described. Emphasis is put on the chemical reactions and the importance of providing enantiopure synthons. In particular kinetics of resolution is in focus. Among the topics covered are enzyme classification, structure and mechanism of action of enzymes. Examples are given on the use of hydrolytic enzymes such as esterases, proteases, lipases, epoxide hydrolases, acylases and amidases both in aqueous and low-water media. Reductions and oxidations are treated both using whole cells and pure enzymes. Moreover, use of enzymes in sngar chemistiy and to prodnce amino acids and peptides are discnssed. 

Enzyme catalysed hydrolysis of racemic epoxides is interesting from a practical point of view. This reaction is catalysed by epoxide hydrolases (EHs, EC 3.3.2.3) (Archelas and Furstoss, 1998). Mammalian EHs are the most widely studied and they are divided into five groups among which the soluble (cytosolic) epoxide hydrolases (sEH) and microsomal epoxide hydrolases (mEH) are best charactelised. The mechanism of sEH from rat starts with a nucleophilic attack by Asp333 on a carbon of the epoxide (usually the least hindered one) to form a glycol monoester intermediate which is stabilised by an oxyanion hole. A water molecule activated by His523 releases the 1,2-diol product. An... 

CYP450s include steroid hormones and lipid-soluble drugs (Brown, 2001). Oxidative reactions frequently lead to the formation of highly reactive epoxides. These toxic metabolites are usually detoxified rapidly by phase II conjugation or other mechanisms, such as microsomal epoxide hydrolases (Pineiro-Carrero and Pineiro, 2004 Watkins, 1999). 

Figure 17. A possible mechanism for the hydration of phcnanthrene 9,10-oxidc by epoxide hydrolase. The pscudo-sccond-order rate constant for the enzyme-catalyzed reaction has been estimated to be 10 times greater than the correspond-...

When racemic aryl glycidyl ethers were subjected to aminolysis in aqueous buffer catalyzed by hepatic microsomal epoxide hydrolase from rat, the corresponding (S)-configurated amino-alcohols were obtained in 51-88% ee 131. On the other hand, when azide was employed as nucleophile for the asymmetric opening of 2-methyl-1,2-epoxyheptane in the presence of an immobilized crude enzyme preparation derived from Rhodococcus sp., which contains an epoxide hydrolase activity, the reaction revealed a complex picture 1321. The (S)-epoxide from the racemate was hydrolyzed (as in the absence of azide), and the less readily accepted (i )-enantiomer was transformed into the corresponding azido-alcohol (ee >60%). Although at present only speculations can be made about the actual mechanism of both the aminolysis and azidolysis reaction, in both cases it was proven that the reaction was catalyzed by a protein and that no reaction was observed in the absence of biocatalyst... 

Computational approaches to evaluate different mechanistic proposals for an enzyme have made great strides in the past 10 years. The chapter by Hopmann and Himo describe one such approach and its application to three different enzymatic reactions involving the transformation of an epoxide. The procedures and parameters to make a model of the active site are presented first and are followed by discussions of limonene epoxide hydrolase, soluble epoxide hydrolases, and haloalcohol dehalogenase. The results generally support the currently accepted mechanism for each enzyme but provide new insights into their regioselectivities. 

Scheme 6 Epoxide hydrolysis mechanism of soluble epoxide hydrolase (residue numbering as in human sEH). Step A is referred to as the alkylation half-reaction while steps B and C comprise the hydrolytic half-reaction.

Hydrolysis of epoxides, esters, amides, and related structures is an important biotransformation reaction that limits the therapeutic activity of many drugs and generates therapeutically active drugs from prodmg structures. In a few cases, hydrolytic reactions can generate a toxic structure. Epoxide hydrolases and esterases are members of the a/(3 hydrolase-fold family of enzymes (Morisseau and Hammock, 2005 Satoh and Hosokawa, 2006). Although their substrate specificities are radically different (e.g., lipids, peptides, epoxides, esters, amides, haloalkanes), their catalytic mechanisms are similar. All of these enzymes have an active site catalytic triad composed of a nucleophilic serine or cysteine residue (esterases/amidases), or aspartate residue (epoxide hydrolases) to activate the substrate, and histidine residue and glutamate or aspartate residues that act cooperatively in an acid—base reaction to activate a water molecule for the hydrolytic step. 

The mechanism of epoxide hydrolase-catalyzed hydrolysis has been elucidated from microsomal epoxide hydrolase (MEH) and bacterial enzymes to involve the trans-antiperiplanar addition of water to epoxides and arene oxides to give vicinal diol products. In general, the reaction occurs with inversion of configuration at the oxirane carbon atom to which the addition takes place and involves neither cofactors nor metal ions [565]. Two types of mechanism are known (Scheme 2.87). 

---