The influence of substrate peptide length on human beta-tryptase specificity.
Combinatorial chemistry approach was applied to design chromogenic substrates of human beta-tryptase. The most active substrate, Ala-Ala-Pro-Ile-Arg-Asn-Lys-ANB-NH(2), was selected from among over 9 million heptapeptides. The amide of 5-amino-2-nitrobenzoic acid (ANB-NH(2)) attached at the C-terminus served as a chromophore. In order to determine the optimal length of the tryptase substrate, a series of N-terminally truncated fragments of this substrate was synthesized. Pro-Ile-Arg-Asn-Lys-ANB-NH(2), with the determined value of the specificity constant (k(cat)/K(M)) above 9 x 10(6) M(-1) s(-1), appeared to be the most specific substrate of tryptase. This substrate was twice as active as the parent heptapeptide substrate. We postulate that the optimal size of the pentapeptide substrate for the interaction with human beta-tryptase is associated with the unique structure of this proteinase, comprising four almost identical monomer subunits arranged in a square flat ring with its substrate pockets faced inside, forming a tetramer with a central pore that can be penetrated by this short peptide.
Spichalska
Bozena
B
Bioorganic Chemistry Department, Faculty of Chemistry, University of Gdańsk, Sobieskiego 18 80-952 Gdańsk, Poland.
Lesner
Adam
A
Wysocka
Magdalena
M
Sledź
Marcin
M
Legowska
Anna
A
Jaśkiewicz
Anna
A
Miecznikowska
Hanna
H
Rolka
Krzysztof
K
D002627:Chemistry, Physical; D020650:Combinatorial Chemistry Techniques; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D019151:Peptide Library; D013379:Substrate Specificity; D053802:Tryptases / Q000737:chemistry* / Q000187:drug effects
D009842:Oligopeptides; D019151:Peptide Library; D053802:Tryptases