Peptide bond formation mediated by substrate mimetics. Structure-guidedoptimization of trypsin for synthesis.
Substrate mimetics are excellent tools for protease-mediated peptide synthesis that enable the coupling of peptides independently of the primary specificity of the enzyme without undesired cleavages of the newly formed peptide bonds. However, the synthetic utility of this beneficial approach is limited to reactions with nonspecific amino-acid-containing peptides while the coupling of specific ones leads to unwanted cleavages due to the native proteolytic activity of the biocatalyst. This paper reports on the use of site-directed mutagenesis to design trypsin variants with decreased cleavage activity. Starting from the variant D189S, which is known for its low proteolytic potential, Ser189 and Ser190 were exchanged for Ala to further repress the inherent amidase activity of trypsin D189S. The effect of mutations was analysed by model synthesis reactions using specific amino-acid-containing peptides and substrate mimetics as the reactants. Finally, computer-assisted protein-ligand docking studies were performed to get closer insight into the molecular basis of the experimental results.
Grünberg
R
R
Department of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany.
Domgall
I
I
Günther
R
R
Rall
K
K
Hofmann
H J
HJ
Bordusa
F
F
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D010455:Peptides; D014357:Trypsin