Mechanistic Studies on ADAMTS13 Catalysis

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• 作者：Enrico Di Stasio ; Stefano Lancellotti ; Flora Peyv ; i ; Roberta Palla ; Pier Mannuccio Mannucci ; Raimondo De Cristofaro
• 刊名：Biophysical Journal
• 出版年：2008
• 出版时间：1 September 2008
• 年：2008
• 卷：95
• 期：5
• 页码：2450-2461
• 全文大小：292 K

文摘

The zinc-protease a disintegrin-like and metalloprotease with thrombospondin type I repeats (ADAMTS13) cleaves the Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ peptide bond of von Willebrand factor (VWF), avoiding the accumulation of ultra large VWF multimers. Hydrolysis by ADAMTS13 of a VWF analog (Asp¹⁵⁹⁶-Arg¹⁶⁶⁸ peptide, fluorescence energy transfer substrate [FRETS]-VWF73) was investigated by a fluorescence quenching method (FRETS method) from 15°C to 45°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 Co²⁺-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 Met¹⁶⁰⁶-Arg¹⁶⁶⁸ peptide product acted as hyperbolic mixed-type inhibitor of FRETS-VWF73 hydrolysis. Asp¹⁶⁵³, Glu¹⁶⁵⁵, Glu¹⁶⁶⁰, Asp¹⁶⁶³, together with the hydrophilic side chain of Thr¹⁶⁵⁶ 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 Met¹⁶⁰⁶-Arg¹⁶⁶⁸ 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 Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ peptide bond for the cleavage by the zinc-aquo complex in the active site.