An Enzymatic Approach to the Synthesis of Peptide Thioesters: Mechanism and Scope

Abstract

[a] Proteases have long been used as biocatalysts for organic transformations.[1] The greatest synthetic value of a protease lies perhaps with its ability to catalyze reverse hydrolysis and other nonhydrolytic reactions in which a nucleophile other than water is engaged in the formation of the desired product. For a serine or cysteine protease, the nucleophile acts as the acceptor of the acyl group from the acyl-enzyme intermediate in a reaction that is made under either thermodynamic or kinetic control.[2] Although a number of useful strategies have been developed and applied successfully to the synthesis of peptides [3][4][5][6][7] and peptide esters, [8] enzymatic synthesis of peptide thioesters has remained a challenge. Herein we demonstrate that subtiligase, a designer peptide ligase derived from the serine protease subtilisin, [6] is a viable catalyst for the thiolysis of peptide esters to form peptide thioesters, an important class of compounds that have seen increased use in the total synthesis of proteins. [9,10] Catalytic peptide ester-to-thioester transesterification by any serine or cysteine protease may appear straightforward, as thiolysis of the acyl-enzyme (acyl-E) intermediate would lead to direct formation of a peptide thioester product in a manner similar to aminolysis (Scheme 1). However, unlike aminolysis of a peptide ester in which a more stable peptide bond is formed, thiolysis forms a more labile thioester bond, which can be quickly hydrolyzed under normal enzyme catalysis conditions. Owing to this and the risk of cleavage of the susceptible peptide bonds notwithstanding, it would be difficult to use a natural serine or cysteine protease for such a transesterification reaction. In fact, our attempts to use subtilisin and papain for this purpose were unsuccessful. We therefore turned our attention to subtiligase. Subtiligase is an engineered double mutant of subtilisin, in which the active site residue Ser 221 is changed to Cys, and the Pro 225 residue to Ala. [6] It has all the structural features of a cysteine protease but lacks the normal amidase activity of the latter while retaining significant esterase activity to catalyze the aminolysis of a suitable peptide ester for peptide ligation.[6] With its decreased hydrolase activity, subtiligase generates a longer-lived acyl-E intermediate, which would make its capture possible by a thiol nucleophile. Because thiols are better nucleophiles than water, a kinetically controlled reaction would favor thiolysis over hydrolysis, and a less hydrolytically active subtiligase would make it possible to isolate the thioester product before it is hydrolyzed by the enzyme.To test our hypothesis, we produced a subtiligase variant that has a His 6 tag at its C terminus to facilitate purification. The presence of this His 6 tag did not affect the enzymatic activity, as tested in a model ligation reaction between the glycolate ester peptide, Ac-His-Ala-Ala-Pro-Phe-glc-Phe-Gly-NH 2 , and the peptide nucleophile, H-Ala-Phe-Ala-NH 2 , which respectively re...

Metabolic Regulation of Protein N-Alpha-Acetylation by Bcl-xL Promotes Cell Survival

“…To this end, we developed a selective biotin labelling method using an engineered protein ligase, termed subtiligase (Abrahmsen et al, 1991; Tan et al, 2007) that detects non-acetylated N-termini of endogenous proteins. This approach was used to capture unmodified protein N-termini resulting from caspase mediated cleavage during apoptotic cell death (Mahrus et al, 2008).…”

“…The expression construct for subtiligase was prepared using the plasmid pBS42 (ATCC 37279) according to published procedures (Wells et al, 1983) except that a His 6 tag was added to the C-terminus (Tan et al, 2007). The gene of the subtiligase variant contains point mutations S221C, P225A, M124L and S125A on wild-type subtilisin BPN′.…”

“…The recombinant protein was expressed in B. subtilis RIK1285, which is deficient in the production of neutral and alkaline proteases. Purification of subtiligase was performed as previously described (Abrahmsen et al, 1991) except that a Co 2+ affinity chromatography step was used instead of ion-exchange chromatography (Tan et al, 2007). The affinity-purified subtiligase was desalted using a PD-10 column (Amersham) with deionized H 2 O. Aliquots of the desalted enzyme solution were flash-frozen, lyophilized and stored at −80°C until utilized.…”

Peptide asparaginyl ligases—renegade peptide bond makers

“…The stability of the subtiligase against heat, alkali, and organic solvents was further enhanced by incorporating five other mutations [69], which allowed the enzyme to catalyze reactions in denaturants. An interesting application by Liu and coworker [70][71][72][73] was to exploit the significant increase in esterase activity and reduced hydrolase activity of modified subtilisin to prepare peptide thioesters and thioacids, which served as the substrates for sequential ligation of unprotected peptides and proteins. Recently, Wells and coworkers [74] used the vast proteome-derived peptide libraries to modify N-terminal substrate scope to allow rapid pairing of selected subtiligase mutants for ligation.…”

Making Circles: Recent Advance in Chemical and Enzymatic Approaches in Peptide Macrocyclization

“…Ligation site within a helix should be avoided during experimental design. Subtiligase mediates fast reactions of protein terminal labelling [100], cyclization [101], and preparation of peptide thioester and thioacid [102,103]. Due to the large binding surface involving seven sites, the minimal length of substrate for macrocyclization requires 12 residues [101].…”