Intermediates enzymes with oxyanion holes

Enzymes with oxyanion holes are now known to catalyze a wide range of reactions with substrates that have a carbonyl moiety. The examples discussed in this chapter include thioesters, oxygen esters, peptides, and ketones (Figure 4.1). Two classes of high-energy intermediates with oxyanions are generated in these reactions (Table 4.3), a tetrahedral intermediate and an enolate. These reactions are... 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

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... 

NH groups is apparently good only for the tetrahe-drally bonded oxyanion intermediate,262 a structure thought to be close to that of the transition state. The importance of the oxyanion hole for catalysis has been supported by theoretical calculations245 and by the study of mutant enzymes.263 In subtilisin, in which a side chain of asparagine forms part of the oxyanion hole, replacement of Asn with the isosteric Leu causes /ccat to fall by a factor of about 200 while Km is unaffected 264 It is also of interest that thiono ester substrates, in which the C=0 of an oxygen ester has been replaced by C = S, bind with normal affinity to chymotrypsin but are not hydrolyzed at significant rates.265... 

Figure 12-15 Schematic drawing of the active site of a cysteine protease of the papain family with a partial structure of an acyl-enzyme intermediate in green. The thiolate-imidazolium pair of Cys 25 His 159 lies deep in the substrate-binding cleft and bridges an interface between two major structural domains, just as the Ser His pair does in serine proteases (Fig. 12-10). This may facilitate small conformational changes during the catalytic cycle. Asn 175 provides a polarizable acceptor for positive charge, helping to stabilize the preformed ion pair, and allows easy transfer of an imidazolium proton to the product of substrate cleavage. The peptide NH of Cys 25 and the side chain of Gin 19 form an oxyanion hole.

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.

Circiunstantial support for this mechanism was supplied by the fact that A-tosyl-Phe-CMK, a specific inhibitor of chymotrypsin, did not react with anhydrochymotrypsin [104]. Although both X-ray crystallographic and NMR studies supported the alkylated hemiketal as the structure of the inhibited enzyme, those studies did not prove whether alkylation or hemiketal formation oecurred first [105, 98]. Carbon-13 NMR studies were also used to determine the pKa (7.88-8.1) of the hemiketal hydroxyl and this finding provided the first evidence that serine proteinases could stabilize the ionized form of the alkylated hemiketal, via hydrogen bonds in the oxyanion hole [106,107]. A series of more recent papers has confirmed that hemiketal formation precedes the alkylation step and has shown that the initial, reversible part of the interaction is made up of two discrete stages (a) formation of a Michaelis complex, followed by (b) hemiketal formation [102, 108]. The requirement of an intermediate hemiketal may mean that chloromethyl ketone (CMK) inhibitors should be considered as transition-state [109] analogue inhibitors (see diseussion in seetion on Aldehydes). 

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