Abstract
Proteases have evolved to mediate the hydrolysis of peptide bonds but may perform transpeptidation in the presence of a proper nucleophilic molecule that can effectively compete with water to react with the acyl-enzyme intermediate. There have been several examples of protease-mediated transpeptidation, but they are generally inefficient, and little effort has been made to systematically control the transpeptidation activity of other proteases with good nucleophiles. Here, we developed an on-bead screening approach to find a probe that functions efficiently as a nucleophile in the protease-mediated transpeptidation reaction, and we identified good probes for a model protease DegP. These probes were covalently linked to the C-termini of the cleaved peptides in a mild condition and made the selective enrichment of ligated peptides possible. We suggest that good nucleophilic probes can be found for many other proteases that act via acyl-enzyme intermediates, and these probes will help characterize their substrates.
Highlights
Proteases, one of the largest groups of enzymes in all organisms, are involved in diverse cellular processes by irreversibly cleaving peptide bonds
Proteases have evolved to catalyze the efficient hydrolysis of peptide bonds, those making acyl-enzyme intermediates may mediate transpeptidation or peptide ligation if amine nucleophiles react with the acyl-enzyme intermediate and induce aminolysis instead of hydrolysis (Figure 1)
When DegP-containing OBOC libraries were mixed with a model substrate carrying an N-terminal biotin, the beads with good nucleophiles were labeled with biotin by transpeptidation and detected by blue precipitates on beads which were generated by the reaction of biotin-bound streptavidin-alkaline phosphatase (SAAP) with its substrate, 5-bromoMolecules 2018, 23, 2109
Summary
One of the largest groups of enzymes in all organisms, are involved in diverse cellular processes by irreversibly cleaving peptide bonds. Molecules 2018, 23, x FOR PEER REVIEW are generally very inefficient processes in which hydrolysis dominates It is unknown whether it is possible to find a good nucleophile that will efficiently react with acyl-enzyme intermediates under mild. We developed a strategy to find a good nucleophilic probe for protease-mediated transpeptidation using peptide libraries and on-bead screening This method led to the identification that are generated situ, and they areto generally very inefficient processesforming in whichacyl-enzyme hydrolysis of probes that areinefficiently ligated the C-termini of the peptides dominates. It is unknown whether it is possible to find a good nucleophile that will intermediates with the model DegP protease.
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