Covalent catalysis acyl-enzyme intermediate

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. 

Covalent enzyme catalysis involves the formation of a transient covalent bond between an enzyme and its substrate. Below are the general structures of commonly encountered so-called acyl-enzyme intermediates and other covalent derivatives. 

Figure 9.5. Covalent Catalysis. Hydrolysis by chymotrypsin takes place in two stages (A) acylation to form the acyl-enzyme intermediate followed by (B) deacylation to regenerate the free enzyme.

The covalent catalysis progresses by formation of an impermanent acyl-enzyme intermediate. It and the (3-lactam binding involve the same serine residue of the enzyme s active site. The three-dimensional features of a penicillin-sensitive D-alanyl-carboxypeptidase-transpeptidase and exact binding site of a penicillin and cephalosporin have been established by X-ray crystallographic studies. One of the results of these experiments is to provide physical support for Tipper and Strominger s (1965) original hypothesis that P-lactam antibiotics and the normal substrate bind on the same locus of the transpeptidase. 

Many enzymes form covalent bonds with their substrates during catalysis, such as the acyl-enzyme intermediate in carboxyl ester hydrolysis (Scheme 2.1) or the glycol monoester intermediate in epoxide hydrolysis (Scheme 2.85). Despite the covalent enzyme-substrate bond, such species are metastable and should be regarded as activated intermediates . Some enzymes utilize cofactors, such as... 

Several bacterial penicillin-binding proteins have been shown to be serine enzymes (Section 2). P-Lactamases are efficient and clinically important enzymes which play an important part in bacterial resistance to the normally lethal action of P-lactam antibiotics. A major class of P-lactamases are also serine enzymes that function by covalent catalysis with the intermediate formation of an acyl-enzyme (Knott-Hunziker et al., 1982 Cohen and Pratt, 1980 Fisher et al., 1980 Anderson and Pratt, 1983 Cartwright and Fink, 1982, Joris et al., 1984). The P-lactamase catalysed hydrolysis of a penicillin thus proceeds by formation of an acyl-enzyme which is an a-penicilloyl ester of a serine residue. The mechanism of reaction of penicillin with alcohols is therefore of obvious relevance, but in addition acyl transfer from nitrogen to oxygen nucleophiles is of current interest. 

Leaving group departure is general-acid-catalyzed by the imidazolium created in the first step (step 2). This forms an acyl-enzyme intermediate. Hence, this represents a form of covalent catalysis, where the high effective molarity of the serine nucleophile further enhances the rate. Water then comes into the active site, and then all the steps just discussed are simply repeated with water as the nucleophile. 

Rossi and Bernhard (1971) attempted to show the close relation between catalysis and the conformational variations of an enzyme molecule. Their study indicated that the rate of nucleophile-catalyzed hydrolysis of an acyl chymotrypsin (the o-/ -(3-indole)-acryloyl]-Ser 195-chymotrypsin), is the same as the rate of denaturation of the acyl enzyme when evaluated from the optical properties of the acyl group in the enzyme. Thus, Rossi and Bernhard (1971) concluded that a covalently modified acyl enzyme-nucleophile complex is an obligatory intermediate in both catalysis and denaturation of this acyl enzyme. 

Covalent intermediates in serine protease catalysis have played a significant and historical role in our understanding of enzyme mechanism. During catalysis the Substrate will pass through at least two covalent intermediates. The- tetrahedral adduct is formed before generation of the acyl enzyme. Trapping of covalent acyl enzymes from specific substrates is very difficult since in this substrate type the... 

Reactions can be accelerated by the formation of intermediates, provided that such intermediates are rapidly formed and broken down. This then provides a new reaction pathway of lower activation energy. Many enzyme-catalyzed reactions proceed via covalent catalysis, involving side chains such as Cys, Ser, Eys. or His acting as nucleophiles in the formation of intermediates such as acyl-enzymes or Schiff bases. Changes in environment... 

The facts of amino and acyl transfer in the course of peptic catalysis, however, do not by themselves prove the covalent nature of the intermediates, such as the acylation of the amion component of the substrate by the enzyme carboxyls (amino-enzyme) or the anhydride intermediates formed by the acyl component of the substrates by the enzyme carboxyls (acyl-enzyme) (Fig.6). 

The side chains of amino acids present a number of nucleophilic groups for catalysis these include RCOO-, R—NH2, aromatic—OH, histidyl, R—OH, and RS. These groups attack electrophilic (electron-deficient) parts of substrates to form a covalent bond between the substrate and the enzyme, thus forming a reaction intermediate. This type of process is particularly evident in the group-transfer enzymes (EC Class 2 see Table 8.1). In the formation of a covalently bonded intermediate, attack by the enzyme nudeophile (Enz-X in Example 8.10) on the substrate can result in acylation, phosphorylation, or glycosylation of the nucleophile. 

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