Chymotrypsin activation

Berezin, I. V., A. V. Levashov, and K. Martinek, On the modes of interaction between competitive inhibitors and the alpha-chymotrypsin active centre , FEBS Lett., 7,20-22 (1970). 

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. 

Although all pancreatic enzymes are inactivated during intestinal transit, fecal outputs of several enzymes correlate with pancreatic enzyme secretion. Fecal chymotrypsin activity, which is comparatively stable in the lumen as well as in extracorporal fecal samples, can be measured by a commercially available photometric test kit. When performed on three consecutive days, this test detects severe pancreatic exocrine insufficiency, but sensitivity and specificity are low in mild-to-moderate cases. In addition, the test does not differentiate between porcine and human chymotrypsin, so that pan-creatin supplements need to be discontinued 5 days prior to the test. For this reason, however, the test is able to monitor a patient s compliance in severe pancreatic insufficiency appar-endy refractory to enzyme treatment. Patients... 

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. 

Figurel.10. Stereo diagram of the acyl moiet of the spin-labeled tryptophanyl-acylenzyme reaction intermediate of a-chymotrypsin. Active site residues close to the acyl moiety are labeled (Makinen et al., 1998). Reproduced with permission.

Fig. 2.13 illustrates the electrostatic effects in transition state in enolase reaction (Larson et al., 1996). During this reaction a proton is removed by Lys-345 from C-2 of 2-phosphoglycerate to give an enolyzed, charged intermediate. This intermediate is stabilized by electrostatic interaction with five positive charges supplied by two Mg+2 ions and a protonated lysine. The 10-11 electrostatic interactions were found in the transition state of formate dehydrogenase and carbamoyl synthetase (Bruice and Benkovic, 2000) Another example of multifunctional interactions during enzymatic reactions in intermediate is the X-ray structure of tetrahedral intermediate in the chymotrypsin active site (Fig. 1.1). 

I. Willner, M. Lion-Dagan, S. Rubin, J. Wonner, F. Effenberger, and P. Bauerle, Photoregulation of a-chymotrypsin activity in organic media effects of bioimprinting, Photochem. Photobiol., 59, 491-496 (1994). 

Application and Principle This procedure is used to determine chymotrypsin activity in chymotrypsin preparations derived from purified extracts of porcine or bovine pancreas. 

Chymosin (Aspergillus niger var. awamori, Escherichia coli K-12, and Kluyveromyces marxianus, each microorganism containing a calf prochymosin gene), 786, (S3)20 Chymotrypsin, 786, (S3)18 Chymotrypsin Activity, 793 Cinene, 518 1,8-Cineol, 496 Cineole, Percentage of, 818 Cinnamal, 468 Cinnamaldehyde, 468, 611 Cinnamic Acid, 468, 565, 612, (S3)66 Cinnamic Alcohol, 470 Cinnamic Aldehyde, 468 Cinnamon Bark Oil, Ceylon Type, 101, 578... 

Catalase Activity, 791 Cellulose Activity, 791 Chymotrypsin Activity, 793 Diastase Activity (Diastic Power), 793 a-Galactosidase Activity, 794 [i-Glucanasc Activity, 795 Glucoamylase Activity... 

Chymotrypsinogen and chymotrypsin Activation of zymogen —> Tyr A-spectrum autolysis or urea treatment of enzyme —> Try A-speotrum Chervenka (1959)... 

There appears to be no single pattern describing the hydration dependence of enzyme activity. Lysozyme activity is correlated with the unfreezing of surface motion at 0.25 A and also with the onset of surface percolation. There are changes in activity above 0.38 A, such as the changes found for rotational motion of TEMPONE. The hydration threshold for chymotrypsin activity, at 0.12 A, is substantially lower than that for lysozyme. A correlation with percolation is an attractive, but untested, possibility. 

Blevins and Tulinsky (1985) suggested two functions for the solvent at the chymotrypsin active site (1) solvation of the Asp—His—Ser catalytic triad, and (2) a guiding effect on the substrate in formation of the enzyme-substrate complex, provided by several waters at the end of the specificity site. X-Ray diffraction results have suggested a role of active-site water in determining the kinetics or equilibria of substrate binding for other proteins (Section IV). 

When racemic 173 (R = Me) was hydrolyzed in the presence of chymo-trypsin, the resulting optically active acid 173 (R = H) exhibited an ORD absorption spectrum characteristic of L-phenylalanine the starting ester possessed an axially oriented carbomethoxy group.338-362 Atropisomerism and conformational asymmetry of a precisely definable nature in a substrate are therefore recognized by chymotrypsin. X-ray diffraction studies confirmed that the chymotrypsin-active isomer has an axial ester moiety in the solid state, and that the ester mutarotates in solution to a CD-inactive isomer, whose ester group is in the equatorial position.363... 

The NBT-PABA test of pancreatic function is based on the hydrolysis, by chymotrypsin, of a synthetic tripeptide— N-benzoyl-l-tyrosyl-p-aminobenzoic acid. The tripeptide, variously called NBT-PABA, BTP, or bentiromide, is administered orally together with a test meal to stimulate pancreatic secretion. BTP is specifically hydrolyzed by chymotrypsin in the duodenum to release PABA, which is subsequently absorbed in the intestinal tract and metabolized in the liver to hippurate and to PABA glucuronide and PABA acetylate. These arylamines are then excreted by the Iddney. In the presence of low chymotrypsin, as found in pancreatic insufficiency, less peptide is hydrolyzed, and therefore less chromogen is excreted in the urine or found in serum. Thus the amount of PABA detected in serum or urine is an indirect measure of chymotrypsin activity in duodenal content. 

Overall, protein suspensions and solutions react similarly to the hydropho-bicity of the solvent. For example, suspensions of ribonuclease, chymotrypsin and lysozyme in nonane, butanol or DMF demonstrated increased structural stability over aqueous conditions (Volkin et al., 1991). The solvents were categorized as non-polar (nonane), polar (butanol) or dipolar (DMF). Thermal stability increased with increasing hydrophobicity and apolarity. Similarly, chymotrypsin activity decreased with decreasing hydrophobicity, consistent with the hypothesis that hydrophilic solvents can more effectively remove bound water than can hydrophobic solvents (Zaks and Klibanov, 1988a). For example, the solubility of water in hydrophobic solvents (Log P < 2.0) was > 0.4 wt %, while the solubility of water in hydrophobic solvents (Log P > 4.0) was < 0.4... 

Figure 13 Phenyl hippurate substrate—chymotrypsin active site complex. Dashed lines show distance constraints assigned for docking.

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