Enzymatic coupling of specific peptides at nonspecific ligation sites: effect of Asp189Glu mutation in trypsin on substrate mimetic-mediated reactions.
Two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. While the latter can be overcome by the use of substrate mimetics achieving peptide bond formation at nonspecific ligation sites, the risk of proteolytic cleavage still remains and hinders the wide acceptance of this powerful strategy for peptide coupling. This paper reports on the effect of the trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mimetics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enzymatic coupling reactions using substrate mimetics as the acyl donor and specific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift in substrate preference from Lys and Arg to the substrate mimetics and, thus, from cleavage to synthesis of peptide bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.
Xu
S
S
Max-Planck Society, Research Unit "Enzymology of Protein Folding", Halle/Saale, Germany.
Rall
K
K
Bordusa
F
F
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers; D018698:Glutamic Acid / Q000737:chemistry*; D006868:Hydrolysis; D018716:Molecular Mimicry; D010455:Peptides / Q000737:chemistry*; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D010455:Peptides; D001224:Aspartic Acid; D018698:Glutamic Acid; D014357:Trypsin