title: Mechanistic studies on ADAMTS13 catalysis.
abstract: The zinc-protease a disintegrin-like and metalloprotease with thrombospondin type I repeats (ADAMTS13) cleaves the Tyr(1605)-Met(1606) peptide bond of von Willebrand factor (VWF), avoiding the accumulation of ultra large VWF multimers. Hydrolysis by ADAMTS13 of a VWF analog (Asp(1596)-Arg(1668) peptide, fluorescence energy transfer substrate [FRETS]-VWF73) was investigated by a fluorescence quenching method (FRETS method) from 15 degrees C to 45 degrees C and pH values from 4.5 to 10.5. The catalysis was influenced by two ionizable groups, whose pK(a) values were equal to 6.41 +/- 0.08 (ionization enthalpy = 32.6 +/- 1.7 kJ/mol) and 4 +/- 0.1 (ionization enthalpy = 3.8 +/- 0.4 kJ/mol), whereas these values were equal to 6 +/- 0.1 and 4.1 +/- 0.1, respectively, in Co(2+)-substituted ADAMTS13. The catalytic process of FRETS-VWF73 hydrolysis showed negative activation entropy (-144 kJ/mol), suggesting that the transition state becomes more ordered than the ground state of the reactants. The k(cat)/K(m) values were not linearly correlated with temperature, as expression of change of the kinetic "stickiness" of the substrate. The Met(1606)-Arg(1668) peptide product acted as hyperbolic mixed-type inhibitor of FRETS-VWF73 hydrolysis. Asp(1653), Glu(1655), Glu(1660), Asp(1663), together with the hydrophilic side chain of Thr(1656) were shown to form a "hot spot" in the VWF A2 sequence, which drives the molecular recognition and allosteric regulation of binding to ADAMTS13. The interaction of the Met(1606)-Arg(1668) region of VWF with ADAMTS13 involves basic residues of the protease and is thus progressively inhibited at pH values >8.50. A molecular model of the FRETS-VWF73 showed that the substrate can fit into the active site only if ADAMTS13 assumes a C-like shape and, interacting with the acidic 1653-1668 region of VWF, properly orients the Tyr(1605)-Met(1606) peptide bond for the cleavage by the zinc-aquo complex in the active site.
authors.affiliation: Institute of Biochemistry and Clinical Biochemistry, Catholic University School of Medicine, Rome, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Di Stasio
authors.forename: Stefano
authors.initials: S
authors.lastname: Lancellotti
authors.forename: Flora
authors.initials: F
authors.lastname: Peyvandi
authors.forename: Roberta
authors.initials: R
authors.lastname: Palla
authors.forename: Pier Mannuccio
authors.initials: PM
authors.lastname: Mannucci
authors.forename: Raimondo
authors.initials: R
authors.lastname: De Cristofaro
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D002384:Catalysis; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human