Chymotrypsin in digestion

Minkowski tried unsuccessfully to prepare an extract of dog pancreas that would reverse the effect of removing the pancreas—that is, would lower the urinary or blood glucose levels. We now know that insulin is a protein, and that the pancreas is very rich in proteases (trypsin and chymotrypsin), normally released directly into the small intestine to aid in digestion. These proteases doubtless degraded the insulin in the pancreatic extracts in Minkowski s experiments. 

The inactive precursors are called trypsinogen, pepsinogen, chymotrypsino-gen, and procarboxypeptidase. These precursors are converted to the active enzymes by hydrolytic cleavage of a few specific peptide bonds under the influence of other enzymes (trypsin, for example, converts chymotrypsinogen to chymotrypsin). The digestive enzymes do not appear to self-destruct, probably because they are so constructed that it is sterically impossible to fit a part of one enzyme molecule into the active site of another. In this connection, it is significant that chymotrypsin attacks denatured proteins more rapidly than natural proteins with their compact structures of precisely folded chains. 

Figure 1. Summary of sequencing of Bombyx PITH. Bars show the peptide sequenced, in which the designations and numbers correspond to the type of derivatization or enzyme used in digestion CAM, carboxamidomethylation C, chymotrypsin E, V8 protease. Arrows indicate the sequence whose carboxyl-terminals are not determined. Small letters show the residues deduced solely from cDNA analysis. Asterisk indicates the site of probable glycosylation.

Proteolytic enzymes, such as the serine proteases, are among the best characterized of all enzymes.They are important in digestive processes because they break down proteins. They each catalyze the same type of reaction, that is. the breaking of peptide bonds by hydrolysis. The crystal structures of several serine proteases have been determined, and the mechanism of hydrolysis is similar for each. The specificity of each enzyme is, however, different and is dictated by the nature of the side chains flanking the scissile peptide bond (the bond that is broken in catalytic mechanism. Chymotrypsin is one of the best characterized of these serine proteases. The preferred substrates of chymotrypsin have bulky aromatic side chains. The crystal structure determination of the active site of chymotrypsin, illustrated in Figure 18.12, has provided much of the information used to elucidate a plausible mechanism of action of the enzyme. In the first step of any catalyzed reaction, the enzyme and substrate form a complex, ES, the Michaelis complex. The hydrolysis of the peptide bond by chymotrypsin involves three amino acid residues,... 

Enzymes, we have said, are proteins that act as enormously effective catalysts for biological reactions. To get some idea of how they work, let us examine the action of just one chymotrypsin, a digestive enzyme whose job is to promote hydrolysis of certain peptide links in proteins. The sequence of the 245 amino acid residues in chymotrypsin has been determined and, through x-ray analysis, the conformation of the molecule is known (Fig. 37.1). It is, like all enzymes, a soluble globular protein coiled in the way that turns its hydrophobic parts inward, away from water, and that permits maximum intramolecular hydrogen bonding. 

Digest 0.8 pg (-1.2 x 10 ) capsids with either 5pg (0.02% final concentration) of trypsin, 1 pg of proteinase K, or 80 pg of a-chymotrypsin in a 25 pi reaction at 37°C for up to 24h. For each serotype, an undigested sample should be included as a control. These proteases are commercially available from Sigma. Add an equal volume of Laemmli sample buffer containing 1% sodium dodecyl sulfate (SDS) and 655 mM p-mercaptoethanol and boil the samples at 100°C for 5 min. 

The zymogen trypsinogen is cleaved to form trypsin by enteropeptidase (a protease, formerly called enterokinase) secreted by the brush-border cells of the small intestine. Trypsin catalyzes the cleavages that convert chymotrypsinogen to the active enzyme chymotrypsin, proelastase to elastase, and the procarboxypeptidases to the car-boxypeptidases. Thus, trypsin plays a central role in digestion because it both cleaves dietary proteins and activates other digestive proteases produced by the panaeas. 

The rate near pH 8 is twice as great as the rate near pH 7 or pH 9. Chymotrypsin helps digest proteins in your intestine. [From M. L. Bender. 

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