Chymotrypsin active site model

Dive, G., D. Dehareng, and J. M. Ghuysen. 1994. Detailed Study of a Molecule in a Molecule N-Acetyl-L-tryptophanamide in an Active Site Model of a-Chymotrypsin. J. Am. Chem. Soc. 116, 2548-2556. 

Dive G, Dehareng D, Ghuysen JM. Detail study of a molecule in a molecule—/V-acctyl-L-tryptophanamide in an active-site model of a-chymotrypsin. J Am Chem Soc 1994 116 2548-2556. 

Cram s group, like that of Lehn, is attempting to imitate enzyme catalysis via kinetic acceleration through host-guest complexation. A series of transacylase mimics have been synthesized [44] which contain the complexing site, the proton transfer catalyst and the nucleophile found in the chymotrypsin active site. Relatively rapid rates of acylation were observed. It is obvious, however, that additional information will have to be acquired and more model compounds synthesized before the mode of action of an enzyme such as chymotrypsin can be clarified. 

A similar concept was used in the development of artificial chymotrypsin mimics [54]. The esterase-site was modeled by using the phosphonate template 75 as a stable transition state analogue (Scheme 13.19). The catalytic triad of the active site of chymotrypsin - that is, serine, histidine and aspartic acid (carboxy-late anion) - was mimicked by imidazole, phenolic hydroxy and carboxyl groups, respectively. The catalytically active MIP catalyst 76 was prepared using free radical polymerization, in the presence of the phosphonate template 75, methacrylic acid, ethylene glycol dimethacrylate and AIBN. The template removal conditions had a decisive influence on the efficiency of the polymer-mediated catalysis, and best results were obtained with aqueous Na2CC>3. 

In our initial research on semisynthetic enzymes, we examined briefly the modification of the serine proteinase a-chymotrypsin, perhaps the best understood of the proteolytic enzymes. A logical choice as a residue for alkylation in the active site of a-chymotrypsin is His-57. However, an examination of a three-dimensional model (Lab Quip) of chymotrypsin in which coenzyme analogs were covalently attached to His-57 suggested strongly that such modifications would block completely the enzyme s active site region and that the probability of new reactions being catalyzed by the modified enzyme would be low. Another possible site of modification of chymotrypsin that could be considered was Met-192. This residue, located on the periphery of the... 

The most promising direction for enzyme modeling is to synthetically mimick the nature of the binding site and the active site in terms of the close similarity of catalytic groups, stereochemistry, interatomic distances and the mechanism of the action of the enzyme. Mimicking of the proton-transfer relay proposed for the mechanism of the action of chymotrypsin is a brilliant example of such work (D Souza and Bender, 1987 and references therein). The miniature organic model of chymotrypsin built on the basis of cyclodextrin and the mechanism of hydrolysis m-tert-butylphenyl acetate is presented in Fig. 6.9. 

Structural Insights, Chymotrypsin A Serine Protease. Work with interactive molecular models to learn more about the structural bases of active site specificity and reactivity, and some of the ways in which active site residues can be identified. 

Chymotrypsin hydrolysis of spin-labeled ester substrates was studied by Electron Nuclear Double Resonance and molecular modeling methods (Wells et al., 1994). The spin-labeled acyl-enzyme was stabilized in low temperatures, and conformations of the substrate in the active site have been assigned from the experiments - both free in solution and in the active site. Conclusions from this study are that significant torsional alteration in the substrate s structrue occurs between its "free" form in solution and its bound form in the active site. The enzyme does not "recognize" the solution structure, but an altered one, that is steieospecifically complementary to the surface of the active site. 

In the recent studies, the enzyme shows that the overall polypeptide fold of chymotrypsin-like serine protease possesses essential SI specificity determinants characteristic of elastase using the multiple isomorphous replacement (MIR) method and refined to 2.3 A resolution Fig. (5). Structure-based inhibitor modeling demonstrated that EFEa s SI specificity pocket is preferable for elastase-specific small hydrophobic PI residues, while its accommodation of long and/or bulky PI residues is also feasible if enhanced binding of the substrate and induced fit of the SI pocket are achieved [Fig. (6) shows the active sites of serine protease]. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. This structure is the first report of an earthworm fibrinolytic enzyme component, a serine protease originated from annelid worm [17]. 

This Highlight is part of an extraordinary story (also a cautionary tale) in the area of biocatalysis. The point of particular interest was the incredible catalytic activity claimed for so-called pepzymes - small synthetic peptides modelled to mimic the active site structures of trypsin and chymotrypsin. One was claimed to hydrolyse a simple peptide (a trypsin substrate) with efficiency comparable to that of the native enzyme. This extraordinary result provoked at least as much scepticism as excitement, and in the following months several groups tried to reproduce the results. They failed, comprehensively. [1,2] Some reasons why this failure came as no surprise were subsequently summarised by Matthews, Craik and Neurath, [3] and by Corey and Corey [4]. The background has been discussed in an Angewandte Review on Enzyme Mechanisms, Models and Mimics. [5]... 

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