Di Stasio Enrico, Lancellotti Stefano, Peyvandi Flora, Palla Roberta, Mannucci Pier Mannuccio, De Cristofaro Raimondo
Institute of Biochemistry and Clinical Biochemistry, Catholic University School of Medicine, Rome, Italy.
Biophys J. 2008 Sep;95(5):2450-61. doi: 10.1529/biophysj.108.131532. Epub 2008 May 23.
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.
锌蛋白酶——具有I型血小板反应蛋白基序的解聚素样金属蛋白酶（ADAMTS13）可裂解血管性血友病因子（VWF）的Tyr(1605)-Met(1606)肽键，避免超大VWF多聚体的积累。通过荧光猝灭法（荧光共振能量转移法）在15℃至45℃以及pH值为4.5至10.5的条件下研究了ADAMTS13对VWF类似物（Asp(1596)-Arg(1668)肽，荧光共振能量转移底物[FRETS]-VWF73）的水解作用。催化作用受两个可电离基团影响，其pK(a)值分别为6.41±0.08（电离焓=32.6±1.7 kJ/mol）和4±0.1（电离焓=3.8±0.4 kJ/mol），而在Co(2+)取代的ADAMTS13中，这些值分别为6±0.1和4.1±0.1。FRETS-VWF73水解的催化过程显示出负的活化熵（-144 kJ/mol），表明过渡态比反应物的基态更加有序。k(cat)/K(m)值与温度并非线性相关，这体现了底物动力学“粘性”的变化。Met(1606)-Arg(1668)肽产物作为FRETS-VWF73水解的双曲线混合型抑制剂。Asp(1653)、Glu(1655)、Glu(1660)、Asp(1663)以及Thr(1656)的亲水性侧链在VWF A2序列中形成一个“热点”，驱动与ADAMTS13结合的分子识别和变构调节。VWF的Met(1606)-Arg(1668)区域与ADAMTS13的相互作用涉及蛋白酶的碱性残基，因此在pH值>8.50时逐渐受到抑制。FRETS-VWF73的分子模型表明，只有当ADAMTS13呈现C样形状并与VWF的酸性1653-1668区域相互作用时，底物才能进入活性位点，并使Tyr(1605)-Met(1606)肽键正确定向，以便活性位点中的锌水络合物进行裂解。
