Serine protease catalysis

James, M.N.G., et al. Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8 A resolution. A model for serine protease catalysis. 

Sprang, S., et al. The three-dimensional structure of Asn ° mutant of trypsin role of Asp ° in serine protease catalysis. Science 237 905-909, 1987. 

S. Sprang, T. Standing, R. J. Fletterick, R. M. Stroud, J. Finer-Moore, N. H. Xoung, R. Hamlin, W. J. Rutter, C. S. Craik, The Three-Dimensional Structure of Asn102 Mutant of Trypsin Role of Asn102 in Serine Protease Catalysis , Science 1987, 237, 905-909. 

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... 

The previous section demonstrated that chiral macrocyclic polyether hosts discriminate in complexation reactions in chloroform solution between enantiomers of amino ester salt guests. With these results can we go one step further and mimic a catalytic site We will now describe the design of a host that upon complexation with a-amino ester salts produces a transition state intermediate corresponding to a transacylation (thiolysis) reaction between the chiral host catalytic group (thiol) and the enantiomeric guest salts (143). However, it should immediately be realized that these model systems mimic only the acylation step encountered in serine protease catalysis. So far no acceleration in rate has been observed for the deacylation step. 

An example of a pseudoirreversible inhibitor has been demonstrated for chymotrypsin (36). This enzyme is a serine protease, and its mechanism of catalysis may be outlined as follows, where or R2 preferentially is a hydrophobic amino acid residue. 

Kraut, J. Serine proteases structure and mechanism of catalysis. Anna. Rev. Biochem. 46 331-358, 1977. 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

FIGURE 7.2. Two alternative mechanisms for the catalytic reaction of serine proteases. Route a corresponds to the electrostatic catalysis mechanism while route b corresponds to the double proton transfer (or the charge relay mechanism), gs ts and ti denote ground state, transition state and tetrahedral intermediate, respectively. 

Catalysis, specific acid, 163 Catalytic triad, 171,173 Cavity radius, of solute, 48-49 Charge-relay mechanism, see Serine proteases, charge-relay mechanism Charging processes, in solutions, 82, 83 Chemical bonding, 1,14 Chemical bonds, see also Valence bond model... 

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

The catalytic mechanism of the subtilisins is the same as that of the digestive enzymes trypsin and chymotrypsin as well as that of enzymes in the blood clotting cascade, reproduction and other mammalian enzymes. The enzymes are known as serine proteases due to the serine residue which is crucial for catalysis (Kraut, 1977 and Polgar, 1987)... 

Kraut, J. (1977) Serine Proteases Structure and Mechanism of Catalysis Ann, Rev. Biochem 46 331-358... 

R. L. Schowen, Structural and Energetic Aspects of Proteolytic Catalysis by Enzymes Charge-Relay Catalysis in the Function of Serine Proteases , in Mechanistic Principles of Enzyme Activity , Eds. J. F. Liebman, A. Greenberg, VCH, New York, 1988, p. 119 — 165. 

Schowen, R.L. (1988). Structural and energetic aspects of protolytic catalysis by enzymes charge-relay catalysis in the function of serine proteases. In Mechanistic Principles of Enzyme Activity, Liebman, J.P. and Greenberg, A. (eds), pp. 119-168. VCH Publishers, New York... 

Crystallographic studies (Blow, 1976) of the structure of the enzyme, enzyme-substrate complexes and enzyme-product complexes have identified a common feature in catalysis by the serine protease enzymes such as a-chymotrypsin. This is the well-known charge-relay system (44), in which... 

In chymotrypsin and other serine proteases the imidazole moiety of histidine acts as a general base not as a nucleophile as is probably the case in the catalysis of activated phenyl ester hydrolysis by (26). With this idea in mind, Kiefer et al. 40) studied the hydrolysis of 4-nitrocatechol sulfate in the presence of (26) since aryl sulfatase, the corresponding enzyme, has imidazole at the active center. Dramatic results were obtained. The substrate, nitrocatechol sulfate, is very stable in water at room temperature. Even the presence of 2M imidazole does not produce detectable hydrolysis. In contrast (26) cleaves the substrate at 20°C. Michaelis-Menten kinetics were obtained the second-order rate constant for catalysis by (26) is 10 times... 

Figure 3.3 (a) Covalent catalysis the catalytic mechanism of a serine protease. The enzyme acetylcholinesterase is chosen to illustrate the mechanism because it is an important enzyme in the nervous system. Catalysis occurs in three stages (i) binding of acetyl choline (ii) release of choline (iii) hydrolysis of acetyl group from the enzyme to produce acetate, (b) Mechanism of inhibition of serine proteases by diisopropylfluorophosphonate. See text for details. 

This reaction encompasses a number of interesting features (general Brpnsted acid/ Brpnsted base catalysis, bifunctional catalysis, enantioselective organocatalysis, very short hydrogen bonds, similarity to serine protease mechanism, oxyanion hole), and we were able to obtain a complete set of DFT based data for the entire reaction path, from the starting catalyst-substrate complex to the product complex. 

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