Mammalian proteins are the targets of a wide range of covalent modification processes. Modifications such as prenylation, glycosylation, hydroxylation, and fatty acid acylation introduce unique structural features into newly synthesized proteins that persist for the lifetime of the protein. Some covalent modifications regulate protein function. The core principle underlying regulatory covalent modification is that, introducing a new covalent bond or cleaving an existing covalent bond alters the identity of the affected enzyme. This “new” enzyme molecule thus can possess properties distinct from those of its precursor. In mammalian cells, a wide range of regulatory covalent modifications occur. Some of these modifications, such as partial proteolysis, in which one or a small handful of peptide bonds within a precursor protein are hydrolyzed, are termed irreversible because it is impossible to reconstruct the precursor form of the protein inside the cell. Hence, once modified the activated proteins retain their new form and properties until they are damaged, disposed, or degraded.

Acetylation, ADP-ribosylation, sumoylation (the attachment of a small, ubiquitin-like modifier or SUMO protein), and phosphorylation are all examples of so-called reversible covalent modifications. In this context, “reversible” refers to capability to restore the modified protein to its modification free precursor form and not the mechanism by which restoration takes place. Thermodynamics dictates that if the enzyme-catalyzed reaction by which the modification was introduced is thermodynamically favorable, simply reversing the process will be rendered impractical by the correspondingly unfavorable free-energy change. The phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by protein kinases, is thermodynamically favored as a consequence of utilizing the high-energy gamma phosphoryl group of ATP. Phosphate groups are removed, not by recombining the phosphate with ADP to form ATP, but by a hydrolytic reaction catalyzed by enzymes called protein phosphatases. Similarly, acetyltransferases employ a high-energy donor substrate, NAD+, while deacetylases catalyze a direct hydrolysis that generates free acetate.

Activation of Chymotrypsin Illustrates Control of Enzyme Activity by Selective Proteolysis

Many secreted proteins are synthesized as larger, functionally inert precursors calledproproteins. If the latent function of the proprotein is catalytic in nature, these precursors are termed proenzymes or, alternatively, zymogens. Transformation of proproteins into their active, functionally competent forms is accomplished by selective, also known as partial, proteolysis involving one or a small handful of highly specific proteolytic clips. Activation of chymotrypsinogen, for example, involves the hydrolysis of four peptide bonds (Figure 1) that divide the original polypeptide into five smaller segments. The largest of these are designated as peptides A, B, and C. In mature, active α-chymotrypsin the A, B, and C peptides are linked together by disulfide bonds while the two small dipeptides are allowed to diffuse away. Note that while residues His 57 and Asp 102 of the catalytically essential charge relay network  reside on the B peptide, Ser 195 resides on the C peptide, brought together in three-dimensional space by conformational changes triggered by the partial proteolysis of chymotrypsinogen.

Fig1. Activation of pancreatic zymogens in the duodenum .

Many Digestive Enzymes Are Stored as Functionally Dormant Proproteins

In their zymogen form, digestive proteases and lipases can be safely stored in the pancreas as they await their excretion into the stomach. On secretion into the digestive tract, these pancreatic hydrolases become activated by a series of selective proteolytic cleavages that are initiated by the conversion of trypsinogen to trypsin through the proteolytic action of enteropeptidase, formerly known as enterokinase, a protease found in the brush border of the duodenum. Once activated by enteropeptidase, trypsin catalyzes the subsequent conversion of numerous other pancreatic zymogens such as chymotryp sinogen, proelastase, kallikreinogen, procarboxypeptidases A and B, prophospholipase A2, and pancreatic prolipase.

Pepsinogen, which is secreted by gastric chief cells, catalyzes its own activation to pepsin. Autoproteolysis is triggered by conformational changes induced on exposure to the acidic pH of upper gastrointestinal tract. In pancreatitis, premature activation of digestive proteases and lipases leads to autodigestion of healthy tissue rather than ingested proteins.

Selective Proteolysis Enables Rapid Activation of the Blood Clotting & Complement Cascades

On wounding or injury, the rapidity with which blood clots are formed can be critical to survival. Synthesis and secretion of the components of the blood clotting and complement cascades as functionally dormant zymogens enable the building blocks for constructing a potentially life-saving blood clot to be prepositioned, ready to be rapidly activated via a series or cascade of selective proteolytic cleavage events. The com ponents of the complement cascade that attacks invading bacteria are also prepositioned in the circulation in the form of proteolytically activated proproteins .