Transition state peptide bond cleavage

Fig. 3.6. Stereoelectronic control of the cleavage of the tetrahedral intermediate during hydrolysis of a peptide bond by a serine hydrolase. The thin lines represent the reactive groups of the enzyme (serine, imidazole ring of histidine) the thick lines represent the tetrahedral intermediate of the transition state. The full circles are O-atoms open circles are N-atoms. The dotted lines represent H-bonds the thick double arrow indicates an unfavorable dipole-dipole interaction [21]. A (R)-configured N-center B (S)-configured N-center.

The structural analysis of the trypsin inhibitor from bovine pancreas (BPTI) in complex with trypsin shows that the inhibitor occupies and blocks the substrate binding pocket in a highly complementary maimer (fig. 2.9). In the trypsin-BPTI complex, the catalytically essential Ser-OH of trypsin contacts a CO group of the inhibitor in a manner very similar to the tetrahedral transition state of amide or ester bond hydrolysis (see fig. 2.9b). The inhibitor can be likened to a pseudo-substrate and, as such, is bound with high affinity. The cleavage of the peptide bond is, however, not possible due to other circumstances, such as the fact that water is prevented from reaching the active site with the inhibitor boimd. 

Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr). The three-dimensional structure of chymotrypsin is shown in Figure 6-18, with functional groups in the active site emphasized. The reaction catalyzed by this enzyme illustrates the principle of transition-state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. 

Asp and His, of the enzyme locate together to help a nucleophilic attack of Ser on the carbonyl carbon of the substrate. The reaction proceeds through a tetrahedral transition state, cleavage of the peptide bond, rapid diffusion of amine moiety leaving acyl-enzyme intermediate followed by the hydrolysis, giving an acid product. 

This moiety may be viewed as a carbon analogue of the transition state in peptide cleavage. The fragment is apparently close enough in structure to such an intermediate as to fit the cleavage site in peptidase enzymes. Once bound, this inactivates the enzyme as it lacks the scissile carbon-nitrogen bond. All five newer HIV protease inhibitors incorporate this structural unit. 

Independent from studies oriented towards the search for better anti-AIDS drugs, compounds of general formula 60 and 61, where R are natural aminoacid residues, were reacted with simple alcohols like methanol. In this way we prepared, with high purity and satisfactory yields, otherwise difficult to obtain phos-phorothioate [87] and phosphorodithioate [88] amidoesters 64 considered as the analogues of transition state for the cleavage of peptide bond by proteases [89]. 

Intensive work worldwide on the design of renin inhibitors has yielded several classes of potent compounds. Most of these inhibitors are transition-state mimetics of the P -P scissile bond (Fig. 2) combined with peptide residues or analogues thereof [1], Much work has been focused on the minimization of the peptide character of renin inhibitors to overcome the drawbacks of substrate-analogous peptides, such as the instability towards enzymatic cleavage, biliary excretion, and low oral bioavailability. In the course of our studies on renin inhibitors at Roche, we synthesized a series of mimetics incorporating the P4-P3 binding elements [2, 3]. 

These drugs are clearly peptide mimics having a genuine amide portion to the right as drawn, a hydroxamic acid to the left and a core that binds zinc and acts as a transition state mimic for the peptide cleavage. They have a C-C bond instead of the amide linkage in the natural substrate so that the drug cannot be hydrolysed by the metallopeptidase. This is easily seen if we disconnect 71a to show the amino acid tert-leucine 73 and a derivative of succinic acid (butanedioic acid) as the core 72. A similar core is present in trocade 70. We shall discuss the asymmetric synthesis of both core succinates. 

Fig. 8.18 The antibiotic penicilhn inhibits the bacterial enzyme glycopeptide transpeptidase. The transpeptidase is a serine protease involved in cross-hnking components of bacterial cell walls and is essential for bacterial growth and survival. It normally cleaves the peptide bond between two D-alanine residues in a pol5 peptide. Penicillin contains a strained peptide bond within the (3-lactam ring that resembles the transition state of the normal cleavage reaction, and thus penicillin binds very readily in the enzyme active site. As the bacterial enzyme attempts to cleave this penicillin peptide bond, penicillin becomes irreversibly covalently attached to the enzyme s active site serine, thereby inactivating the enzyme.

The reaction mechanisms of some enzymatic reactions are known in detail due to the development of powerful structure elucidation tools, such as X-ray and NMR enzymatic reaction mechanisms are no longer in a black box. One of the most studied hydrolytic enzymes is chymotrypsin, which represents a group of serine proteases. It catalyzes the hydrolysis of peptides to amino acids, and the reaction mechanism is shown in Fig. 10.3. Two amino acid residues of the enzyme. Asp and His, locate together to facilitate nucleophilic attack of Ser on the carbonyl carbon of the substrate. The reaction proceeds through a tetrahedral transition state, cleavage of the peptide bond and rapid diffusion of the amine moiety to leave the acyl-enzyme intermediate, foUowedby hydro lysis to give a carboxyhc acid. 

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