Abstract
Historically, ligase activity by proteases was theoretically derived due to their catalyst nature, and it was experimentally observed as early as around 1900. Initially, the digestive proteases, such as pepsin, chymotrypsin, and trypsin were employed to perform in vitro syntheses of small peptides. Protease-catalyzed ligation is more efficient than peptide bond hydrolysis in organic solvents, representing control of the thermodynamic equilibrium. Peptide esters readily form acyl intermediates with serine and cysteine proteases, followed by peptide bond synthesis at the N-terminus of another residue. This type of reaction is under kinetic control, favoring aminolysis over hydrolysis. Although only a few natural peptide ligases are known, such as ubiquitin ligases, sortases, and legumains, the principle of proteases as general catalysts could be adapted to engineer some proteases accordingly. In particular, the serine proteases subtilisin and trypsin were converted to efficient ligases, which are known as subtiligase and trypsiligase. Together with sortases and legumains, they turned out to be very useful in linking peptides and proteins with a great variety of molecules, including biomarkers, sugars or building blocks with non-natural amino acids. Thus, these engineered enzymes are a promising branch for academic research and for pharmaceutical progress.
Highlights
A large number of data on proteases cleaving all sorts of peptide bonds is available in the scientific publications and textbooks
Peptide esters readily form acyl intermediates with serine and cysteine proteases, followed by peptide bond synthesis at the Nterminus of another residue
Established protease-ligases as subtilisin and V8 protease from Staphylococcus aureus, which is trypsin-like with P1–Glu specificity, were used to demonstrate that macromolecular crowding agents, such as polyethylene glycols (PEGs) and dextran enhanced the ligase reaction for the synthesis of triose-phosphate isomerase [72]
Summary
A large number of data on proteases cleaving all sorts of peptide bonds is available in the scientific publications and textbooks. Only a few natural examples are known for true peptide ligases and some proteases, which work both ways, cleaving and/or synthesizing peptide bonds, protease-catalyzed protein splicing was suggested as a new posttranslational modification [8] Such enzyme-dependent processing is distinct from intein splicing. Plant legumains have gained additional interest in recent years, as some members are distinct ligases, which are important in the synthesis of cyclic plant peptides [26] Another special field in which peptide ligation is thought to play a role is the interaction of a variety of proteases with inhibitors that are first cleaved and religated in the active site. Common proteases and optimized, and engineered variants such as trypsiligase and subtiligase can be used as peptide synthetases under nearly physiological conditions or in organic solvents, which increases the tendency for peptide ligation (Figure 1B) [19]
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