Covalent modification activates some enzymes and inactivates others. That is, some enzymes are wholly inactive until they are covalently modified and then begin to function. In other cases, covalent modification acts in the opposite direction, to inactivate otherwise active enzymes. Some such modifications can be reversed; others cannot.

One of the most widespread modifications is phosphorylation or dephosphorylation of various amino acid side chains (e.g., serine, threonine, tyrosine, and histidine). These kinds of modification are most often a part of complex regulatory pathways, frequently under hormonal control. (See kinase cascade).

Another example of covalent enzyme activation is proteolytic cleavage, found in the pancreatic proteases (such as trypsin, chymotrypsin, elastase, and carboxypeptidase). These enzymes are synthesized in the pancreas in an inactive form because if they were active in the pancreas, they would digest the pancreatic tissue. Rather, they are made as slightly longer, catalytically inactive molecules called zymogens (trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase, respectively). The zymogens must be cleaved proteolytically in the intestine to yield the active enzymes (Figure 11.39). If a small amount of protease becomes active in the pancreas, it can have painful or fatal consequences (i.e., acute pancreatitis). The pancreas protects itself from active protease action by synthesis of a protein called the secretory pancreatic trypsin inhibitor. The binding between trypsin and its inhibitor is one of the strongest noncovalent interactions known in biochemistry. The intestinal tissue is protected somewhat from damage by proteases by its glycosylated surface.

The first step is activation of trypsin in the duodenum. A hexapeptide is removed from the N-terminal end of trypsinogen by enteropeptidase,a protease secreted by duodenal cells. This yields active trypsin, which then activates the other zymogens by specific proteolytic cleavages. Trypsin will also activate other trypsinogen molecules in an autocatalytic process. Activation of a few trypsinogens ultimately leads to activation of many trypsins.

Activation of chymotrypsinogen is shown in Figure 11.40. First, trypsin cleaves the bond between arginine 15 and isoleucine 16. Notice that the N-terminal peptide remains attached to the rest of the molecule due to the disulfide bond between residues 1 and 122. The enzyme is activated by the cleavage due to changes in the conformation of the molecule. These include:

Creation of a new, positively charged N-terminal residue at Ile 16;

Salt bridge formation between Ile 16 and Asp 194 (next to the active site); and

Movement of active site residues so that the amino groups of residues 193 and 195 are properly positioned to hydrogen-bond to the substrate oxyanion in the tetrahedral transition state.

Finally, autocatalytic cleavages to remove residues 14-15 and 147-148 from the molecule produce the final, active form of chymotrypsinogen, called -chymotrypsin.