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Molecular mechanism of the interaction between activated factor XIII and its glutamine donor peptide substrate
Author(s) -
PÉNZES K.,
KÖVÉR K. E.,
FAZAKAS F.,
HARAMURA G.,
MUSZBEK L.
Publication year - 2009
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2009.03291.x
Subject(s) - chemistry , glutamine , stereochemistry , peptide , substrate (aquarium) , biochemistry , binding site , active site , enzyme kinetics , dimer , plasmin , enzyme , peptide sequence , amino acid , biology , organic chemistry , ecology , gene
Summary. Background: Activated factor XIII (FXIII), a dimer of truncated A‐subunits (FXIII‐A 2 *), is a transglutaminase that crosslinks primary amines to peptide‐bound glutamine residues. Because in the few natural substrates of FXIII‐A 2 * no consensus sequence could be identified around the reactive glutamine, studying the interaction between individual substrates and FXIII‐A 2 * is of primary importance. Most of the α 2 ‐plasmin inhibitor (α 2 PI) molecules become truncated by a plasma protease, and the truncated isoform (N1‐α 2 PI) is an important substrate of FXIII‐A 2 *. The crosslinking of N1‐α 2 PI to fibrin plays a major role in protecting fibrin from fibrinolysis. Methods: We studied the interaction of FXIII‐A 2 * with its dodecapeptide glutamine donor substrate, N1‐α 2 PI(1–12), the sequence of which corresponds to the N‐terminal sequence of N1‐α 2 PI. Kinetic parameters for N1‐α 2 PI(1–12) and for its truncated or synthetic mutants were determined by a spectrophotometric assay. The interaction of N1‐α 2 PI(1–12) with FXIII‐A 2 * was investigated by proton nuclear magnetic resonance (NMR) and saturating transfer difference (STD) NMR. Results and Conclusions : Kinetic experiments with peptides in which the Asn1 residue was either truncated or replaced by alanine and proton NMR analysis of the FXIII‐A 2 *–N1‐α 2 PI(1–12) complex demonstrated that Asn1 is essential for effective enzyme–substrate interaction. Experiments with C‐terminally truncated peptides proved that amino acids 7–12 are essential for the interaction of N1‐α 2 PI(1–12) with the enzyme, and suggested the existence of a secondary binding site on FXIII‐A 2 *. Hydrophobic residues, particularly Leu10 and the C‐terminal Lys12, seemed to be especially important in this respect, and direct interaction between hydrophobic C‐terminal residues and FXIII‐A 2 * was demonstrated by STD NMR.