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 superp otent analog of human growth hormone releasing factor, [desNH(2)Tyr(1) ,D-Ala(2),Ala(15)]-GRF(1-29)-NH2 (4), from the precursor, [Ala(15,29)] GRF(4-29)-OH (1). C-Terminal amidation of 1 to form [Ala(15)]-GRF(4-2 9)-NH2 (2) was achieved by carboxypeptidase-Y-catalyzed exchange of Al a(29)-OH for Arg-NH2. The target analog 4 was then obtained by acylati on of segment 2 with desNH(2)Tyr-D-Ala-Asp(OH)-OR (3) (R = CH3CH2- or 4-NO2C6H4CH2-) catalyzed by the V8 protease. In this paper we report o n the use of the recently isolated Glu/Asp-specific endopeptidase (GSE ) from Bacillus licheniformis, which is shown to be an efficient catal yst for the segment condensation of 2 and 3. GSE is more stable than t he V8 protease under the conditions employed (20% DMF, pH 8.2, 37 degr ees C). The extent of conversion of 2 into 4 is limited by proteolyses at Asp(3)-Ala(4) and Asp(25)-Ile(26). However, this proteolysis is vi rtually eliminated by use of the appropriate ester leaving group, R. A systematic study of the kinetics of the GSE-catalyzed segment condens ations of 2 and a series of tripeptide esters, desNH(2)Tyr-D-Ala-Asp(O H)-OR (3) [R=CH3CH2- (3a), CH3- (3b), ClCH2CH2- (3c), C6H5CH2- (3d), 4 -NO2C6H4CH2- (3e)] revealed that the rate of aminolysis versus proteol ysis, and hence the conversion of 2 into 4, increase with increasing s pecificity (V-max/K-m) of GSE for the tripeptide ester. The specificit y varies in the order 3e>3d>3c>>3b>3a and appears to depend on an incr ease in the maximum turnover rate (V-max) with increasing basicity of R. This work demonstrates the coupling of a small peptide segment cont aining unnatural amino acids to the N-terminus of an intermediate-leng th polypeptide, without side-chain protection, by GSE, a potentially l ess costly and more stable alternative to the V8 protease. (C) Munksga ard 1994.