Y-Chymotrypsin

The second group of studies tries to explain the solvent effects on enantioselectivity by means of the contribution of substrate solvation to the energetics of the reaction [38], For instance, a theoretical model based on the thermodynamics of substrate solvation was developed [39]. However, this model, based on the determination of the desolvated portion of the substrate transition state by molecular modeling and on the calculation of the activity coefficient by UNIFAC, gave contradictory results. In fact, it was successful in predicting solvent effects on the enantio- and prochiral selectivity of y-chymotrypsin with racemic 3-hydroxy-2-phenylpropionate and 2-substituted 1,3-propanediols [39], whereas it failed in the case of subtilisin and racemic sec-phenetyl alcohol and traws-sobrerol [40]. That substrate solvation by the solvent can contribute to enzyme enantioselectivity was also claimed in the case of subtilisin-catalyzed resolution of secondary alcohols [41]. 

Search the Enzyme Structure Database for y-chymotrypsin active site (by the aid of the active-site-modified enzyme or active-site-specific inhibitor-enzyme complex) to identify and depict (save pdb file) the catalytic triad of y-chymotrypsin. 

Fig. 19. Stereodrawing of 6-benzyl-3-chloro-2-pyrone bound to the active site of y-chymotrypsin. The structures of the native enzyme (solid lines) and the enzyme-inhibitor complex (clear lines) are overlaid. Reproduced with permission from Ringe et al. (1985).

The third approach to solving this problem (Farber, 1999) involves the preparation of an enzyme-intermediate complex at high substrate concentration for X-ray data collection. Under such a condition active sites in the crystal lattice will be filled with intermediates. Using a combination of flow cell experiments and equilibrium experiments, it is possible to obtain the structure of important intermediates in an enzyme reaction (Bolduc et al., 1995). It was also discovered that some enzyme crystals can be transformed from their aqueous crystallization buffer to nonaqueous solvents without cross-linking the crystals before the transfer (Yennawar et. al., 1995). It is then possible to regulate the water concentration in the active site. The structure of the first tetrahedral intermediate, tetrapeptide -Pro-Gly-Ala-Tyr- in the y-chymotrypsin active site obtained by this method is shown in Fig. 1.1. 

We report the results from a molecular dynamics simulation of the serine protease y-chymotrypsin (y-CT) in hexane. The active site of chymotrypsin contains the "catalytic triad" which consists of Ser-His-Asp. y-CT suspended in nearly anhydrous solvents has been found to be catalytically active. In order for proteins to retain their activity in anhydrous solvents some water molecules are required to be present. These "essential waters" have been suggested to function as a molecular lubricant for the protein. Hexane, having a dielectric constant of 1.89, is a suitable non-aqueous solvent for enzymatic reactions. The low dielectric constant of hexane allows it to not compete with the protein for the essential water and allows enzymes to retain their catalytic activity. y-CT in hexane is thus an ideal system to further explore the effect of non-aqueous solvation on protein structure, function and dynamics. 

Immobilization of Enzymes. Enzymes (carboxypeptidase A, B, and Y, chymotrypsin, thermolysin, trypsin, and V -protease), obtained from Sigma were applied directly for immobilization. About 20 mg of each enzyme was dissolved into 0.1N phosphate buffer pH 7.0, and placed into a 10 x 75 mm test tube with 1 g of succinylamidopropyl glass beads. After degassing, 0.02 umole of l-ethyl-3-(3-dimethylamlnopropyl)-carbodilmide (EDC) (Sigma Chemical Co.) was added to the tube which then was sealed with paraffin and rotated at 4°C overnight for simultaneous activation/immobilization. 

As organic solvents are not the natural environment of enzymes, it has been proposed that they may induce changes in the three-dimensional structure of enzymes. The methods developed to study protein structures are difficult to apply to solid enzymes suspended in nonaqueous environments. However, it was possible to show that the structure of subtilisin Carlsberg and y-chymotrypsin is little affected by the passage to organic media (42,43). 

The Proteases and Their Inhibitors.—Much of the successful work on proteolytic enzymes has concerned the serine proteases. The structures of a-chymotrypsin, y-chymotrypsin, chymotrypsinogen, trypsin," and... 

Studies of y-chymotrypsin by Segal et al and of subtilisin by Kraut et al. with chloromethyl ketone analogues of good phenylalanine polypeptide substrates have indicated further similarities in substrate binding and specificity of these enzymes. It has previously been reported that both enzymes contain a serine at the active site which is acylated by ester substrates, a histidine hydrogen-bonded to this serine which is alkylated by active-site directed halogenomethyl ketones, and an aspartate which is buried and hydrogen-bonded not only to the active-site histidine but also in each case to a further serine. 

The studies now reported show that chloromethyl ketone polypeptide inhibitors bind in an antiparallel jS-pIeated sheet fashion to a length of extended backbone, Ser-125—Leu-126—Gly-127 in subtilisin, and Ser-214— Trp 215—Gly 216 in y-chymotrypsin. In each case there are the same geometric relationships of the pleated sheet to the active serine, and glycine residues are involved in j3-pleated sheet hydrogen bonding in both (see Figure 2). In the case of y-chymotrypsin these deductions were made from... 

Figure 2 Atomic model of y-chymotrypsin inhibited by AAAPCK. Only the portion of the enzyme which may interact with the inhibitor is shown...

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