Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase. Influence of the ester leaving group of the acyl donor on yield and catalytic efficiency.
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)
Bongers
J
J
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey.
Liu
W
W
Lambros
T
T
Breddam
K
K
Campbell
R M
RM
Felix
A M
AM
Heimer
E P
EP
D000215:Acylation; D001407:Bacillus / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D004952:Esters / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D017337:Sermorelin / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity
D004952:Esters; C077568:somatotropin releasing hormone (1-29) NH2, desNH2Tyr(1)-Ala(2,15)-; C090714:somatotropin-releasing hormone (4-29)-OH, Ala(15,29)-; D017337:Sermorelin; D013007:Growth Hormone-Releasing Hormone; D010450:Endopeptidases; C075054:glutamic acid-specific endopeptidase, Staphylococcus aureus