Cloning and Molecular Modeling of Duodenase with Respect to Evolution of Substrate Specificity within Mammalian Serine Proteases That Have Lost a Conserved Active-Site Disulfide Bond

Abstract

Mammalian serine proteases such as the chromosome 14 (Homo sapiens, Mus musculus) located granzymes, chymases, cathepsin G, and related enzymes including duodenase collectively represent a special group within the chymotrypsin family which we refer to here as "granases". Enzymes of this group have lost the ancient active-site disulfide bond Cys191-Cys220 (bovine chymotrypsinogen A numbering) which is strongly conserved in classic serine proteases such as pancreatic, blood coagulation, and fibrinolysis proteases and others (granzymes A, M, K and leukocyte elastases). We sequenced the cDNA encoding bovine (Bos taurus) duodenase, a granase with unusual dual trypsin-like and chymotrypsin-like specificity. The sequence revealed a 17-residue signal peptide and two-residue (GlyLys) activation peptide typical for granases. Production of the mature enzyme is apparently accompanied by further proteolytic processing of the C-terminal pentapeptide extension of duodenase. Similar C-terminal processing is known for another dual-specific granase, human cathepsin G. Using phylogenetic analysis based on 39 granases we retraced the evolution of residues 189 and 226 crucial for serine protease primary specificity. The analysis revealed that while there is no obvious link between mutability of residue 189 and the appearance of novel catalytic properties in granases, the mutability of residue 226 evidently gives rise to different specificity subgroups within this enzyme group. The architecture of the extended substrate-binding site of granases and structural basis of duodenase dual specificity based on molecular dynamic method are discussed. We conclude that the marked selectivity of granases that is crucial to their role as regulatory proteases has evolved through the fine-tuning of specificity at three levels--primary, secondary, and conformational.

How Immune Peptidases Change Specificity: Cathepsin G Gained Tryptic Function but Lost Efficiency during Primate Evolution

“…It may be significant that duodenase and cathepsin G (along with chymases and granzyme B-related peptidases) belong to the subset of immune peptidases that form only three disulfide linkages within the catalytic domain (13)—the fewest such linkages known in the extended trypsin/chymotrypsin family. It is proposed that absence of particular disulfide pair (Cys 191 -Cys 220 ) in the vicinity of the active site (and otherwise highly conserved in serine peptidases) allows structural plasticity (46, 52) such that changes in residues lining the pocket are better tolerated than in peptidases with the disulfide pair. This may explain how primate cathepsin G and perhaps duodenase were able to acquire tryptic activity with as little as a single amino acid change, even though changing chymotrypsin into a tryptic enzyme requires exchange of many more residues, including loops (53).…”

Therapeutic targeting of cathepsin C: from pathophysiology to treatment

“…Mast cell chymases: The term "chymase" classically refers to serine proteases of mast cells with chymotrypsin-like ability to cleave peptide and protein targets after aromatic amino acids. Actually, mast cell chymases are less closely related to digestive enzymes like pancreatic chymotrypsin than to immune cell proteases, such as CatG, NE, and lymphocyte granzymes (Ahmad, Bird, & Kaiserman, 2014;Hellman & Thorpe, 2014;Zamolodchikova, et al, 2005). Moreover, substrate preferences of chymases differ from those of chymotrypsin, and some "chymases", having been affected by specificity-altering mutations in the vicinity of the substrate binding pocket, have little or no chymotryptic activity, but possess elastase, Leu-ase, or Met-ase activity, or have become catalytically inactive (Kunori, et al, 2002) (Caughey, et al, 2008).…”

Mast Cell Peptidases

“…In chymotrypsin, for example, mutations are more likely to reduce activity than change specificity, and wholesale changes in the active site are required to change specificity, say, to that of trypsin. The chameleon-like ability of chymases to change specificity may relate in part to lack of a disulfide bond (Cys 191 -Cys 220 ) that constricts and rigidifies the active site of most other serine peptidases of the trypsin-chymotrypsin family (13,21,23,103). The tight embrace of human chymase with its best known endogenous substrate, angiotensin I, is shown in Figure 2, which conveys a sense of how small changes in enzyme topography in the vicinity of the active site can alter binding affinity.…”