Employing mutants to study thrombin residues responsible for factor XIII activation peptide recognition: a kinetic study.
In the last stages of coagulation, thrombin helps to activate Factor XIII. The resultant transglutaminase introduces covalent cross-links into fibrin thus promoting clot stability. To better understand the roles of individual thrombin residues in recognition and hydrolysis of the Factor XIII activation peptide, mutations within thrombin's aryl and apolar binding site were explored. The thrombin mutants W215A, E217A, W215A/E217A, L99A, and I174A were examined through HPLC kinetics against the substrates FXIII (28-41) V34 AP and FXIII (28-41) V34L AP. Several mutants responded differently to FXIII (28-41) V34 AP vs the cardioprotective V34L AP. W215 provides an important platform for binding and directing FXIII APs for proper hydrolysis. Loss of this platform leads to decreases in kinetics, particularly to the kcat of FXIII V34L AP. E217 also plays a supporting role, but the E217A mutation is not as detrimental as W215A. W215A/E217A is unfavorable for both activation peptides and its coupling effect has been characterized. This mutant can readily bind the peptides but cannot orient them for effective hydrolysis. Kinetic studies with I174A indicate that this thrombin residue is more crucial for interactions with the larger V34L AP segment. The L99A mutation causes deleterious effects to binding and hydrolysis of both APs. The V34L, however, is able to partially compensate for the loss perhaps by increasing contact within the aryl and apolar sites. Understanding how specific FXIII and thrombin residues participate in binding and control hydrolysis may lead to the design of coagulation enzymes whose degree of activation and optimal target site can be controlled.
Isetti
Giulia
G
Department of Chemistry, University of Louisville, 2320 South Brook Street, Louisville, Kentucky 40292, USA.
Maurer
Muriel C
MC
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D005176:Factor XIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000378:metabolism*
D005176:Factor XIII; D013917:Thrombin