Probing thrombin's ability to accommodate a V34F substitution within the factor XIII activation peptide segment (28-41).
In blood coagulation, thrombin helps to activate factor XIII (FXIII) by cleaving the activation peptide (AP) at the R37-G38 peptide bond. The common polymorphism V34L yields a FXIII that is more easily activated than the wild type enzyme. Peptides based on the FXIII (28-41) (28TVELQGVVPRGVNL41) sequence serve as an important model system to evaluate the substrate specificity of thrombin and thus how to regulate FXIII activation. Our previous kinetic and nuclear magnetic resonance (NMR) studies have suggested that the P4-P1 amino acids on this FXIII segment provide key anchors to the thrombin active site surface. Furthermore, the most effective amino acid to have at the P4 position is a leucine. In the current work, a peptide containing V34F was examined to probe the ability to accommodate an aromatic residue at this position. Kinetic parameters for thrombin-catalyzed hydrolysis of FXIII AP (28-41) V34F are comparable with that of the wild type V34. One-dimensional proton line-broadening studies reveal that the 34FVPR37 segment encompassing the P4-P1 positions makes the most contact with the thrombin surface. Two-dimensional transferred-nuclear overhauser effect spectroscopy (NOESY) studies indicate that when the peptide is bound to thrombin, the F34 aromatic ring is oriented to promote P4-P2 interactions with P36. This characteristic has been viewed as a hallmark for V34L. An ability to generate this interaction may promote the ability of FXIII AP (28-41) V34F to remain a viable substrate for thrombin.
Isetti
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Department of Chemistry, University of Louisville, 2320 South Brook Street, Louisville, KY 40292, USA.
Maurer
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MC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D004789:Enzyme Activation; D005176:Factor XIII / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; C493000:factor XIII (28-41); C061895:factor XIII activation peptide; D005176:Factor XIII; D013917:Thrombin