Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase. Influence of the ester leaving group of the acyl donor on yield and catalytic efficiency
Por:
BONGERS J., LIU W., LAMBROS T., BREDDAM K., CAMPBELL R.M., FELIX A.M., HEIMER E.P.
Publicada:
1 ene 1994
Resumen:
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°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 eater 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 the rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4, increase with increasing specificity (V(max)/K(m)) of GSE for the tripeptide ester. The specificity varies in the order 3e>3d>3c>>3b>3a and appears to depend on an increase in the maximum turnover rate (V(max)) with increasing basicity of R. This work demonstrates the coupling of a small peptide segment containing unnatural amino acids to the N-terminus of an intermediate-length polypeptide, without side-chain protection, by GSE, a potentially less costly and more stable alternative to the V8 protease.
Filiaciones:
BONGERS J.:
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
LIU W.:
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
LAMBROS T.:
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
BREDDAM K.:
Department of Chemistry, Carlsberg Laboratory, Valby, Copenhagen, Denmark
CAMPBELL R.M.:
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
FELIX A.M.:
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
HEIMER E.P.:
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
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