Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase. Influence of the ester leaving group of the acyl donor on yield and catalytic efficiency.
Bongers. J J; Liu. W W; Lambros. T T; Breddam. K K; Campbell. R M RM; Felix. A M AM; Heimer. E P EP
Key Findings
- GSE is more stable than V8 protease under synthesis conditions (20% DMF, pH 8.2, 37 °C).
- Choosing appropriate ester leaving groups (e.g., ethyl, nitro‑benzyl) virtually eliminates proteolysis at vulnerable Asp sites.
- Higher enzyme specificity (Vmax/Km) for the tripeptide ester correlates with faster desired aminolysis and higher overall conversion to the target peptide.
Practical Outcomes
- For DIY peptide enthusiasts, using GSE with the optimal ester leaving group can improve yields of GRF‑1‑29 analogs and cut side‑reactions, making enzymatic synthesis more practical. However, the approach still requires access to the specific enzyme and organic solvents, so it may be more suited to well‑equipped labs than casual home setups.
Summary
Scientists showed that an enzyme called GSE from Bacillus can stitch together a modified growth‑hormone‑releasing peptide (GRF‑1‑29) more efficiently than the older V8 protease, especially when the right chemical “leaving group” is attached to the donor piece. This reduces unwanted breakdown and boosts the amount of final product.
Abstract
We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), from the precursor, [Ala15,29]-GRF(4-29)-OH (1). C-Terminal amidation of 1 to form [Ala15]-GRF(4-29)-NH2 (2) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala29-OH for Arg-NH2. The target analog 4 was then obtained by acylation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR (3) (R = CH3CH2- or 4-NO2C6H4CH2-) catalyzed by the V8 protease. In this paper we report on the use of the recently isolated Glu/Asp-specific endopeptidase (GSE) from Bacillus licheniformis, which is shown to be an efficient catalyst for the segment condensation of 2 and 3. GSE is more stable than the V8 protease under the conditions employed (20% DMF, pH 8.2, 37 degrees C). The extent of conversion of 2 into 4 is limited by proteolyses at Asp3-Ala4 and Asp25-Ile26. However, this proteolysis is virtually eliminated by use of the appropriate ester leaving group, R. A systematic study of the kinetics of the GSE-catalyzed segment condensations of 2 and a series of tripeptide esters, desNH2Tyr-D-Ala-Asp(OH)-OR (3) [R = CH3CH2- (3a), CH3- (3b), ClCH2CH2- (3c), C6H5CH2- (3d), 4-NO2C6H4CH2- (3e)] revealed that rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4, increase with increasing specificity (Vmax/Km) of GSE for the tripeptide ester.(ABSTRACT TRUNCATED AT 250 WORDS)
Study Information
pubmed
1994