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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.

SUBMITTER: Di Stasio E

PROVIDER: S-EPMC2517044 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Mechanistic studies on ADAMTS13 catalysis.

Di Stasio Enrico E Lancellotti Stefano S Peyvandi Flora F Palla Roberta R Mannucci Pier Mannuccio PM De Cristofaro Raimondo R

Biophysical journal 20080523 5

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 cata ...[more]

PMID: 18502798

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Project description:Exploration of the chemical reaction space of chemical transformations in multicomponent mixtures is one of the main challenges in contemporary computational chemistry. To remove expert bias from mechanistic studies and to discover new chemistries, an automated graph-theoretical methodology is proposed, which puts forward a network formalism of homogeneous catalysis reactions and utilizes a network analysis tool for mechanistic studies. The method can be used for analyzing trajectories with single and multiple catalytic species and can provide unique conformers of catalysts including multinuclear catalyst clusters along with other catalytic mixture components. The presented three-step approach has the integrated ability to handle multicomponent catalytic systems of arbitrary complexity (mixtures of reactants, catalyst precursors, ligands, additives, and solvents). It is not limited to predefined chemical rules, does not require prealignment of reaction mixture components consistent with a reaction coordinate, and is not agnostic to the chemical nature of transformations. Conformer exploration, reactive event identification, and reaction network analysis are the main steps taken for identifying the pathways in catalytic systems given the starting precatalytic reaction mixture as the input. Such a methodology allows us to efficiently explore catalytic systems in realistic conditions for either previously observed or completely unknown reactive events in the context of a network representing different intermediates. Our workflow for the catalytic reaction space exploration exclusively focuses on the identification of thermodynamically feasible conversion channels, representative of the (secondary) catalyst deactivation or inhibition paths, which are usually most difficult to anticipate based solely on expert chemical knowledge. Thus, the expert bias is sought to be removed at all steps, and the chemical intuition is limited to the choice of the thermodynamic constraint imposed by the applicable experimental conditions in terms of threshold energy values for allowed transformations. The capabilities of the proposed methodology have been tested by exploring the reactivity of Mn complexes relevant for catalytic hydrogenation chemistry to verify previously postulated activation mechanisms and unravel unexpected reaction channels relevant to rare deactivation events.

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Mechanistic studies on ADAMTS13 catalysis.

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Mechanistic studies on ADAMTS13 catalysis.

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