Mechanistic studies on ADAMTS13 catalysis
Mechanistic studies on ADAMTS13 catalysis
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DOI：
10.1529/biophysj.108.131532
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发表时间：
2008-09-01
影响因子：
3.4
通讯作者：
De Cristofaro, Raimondo
中科院分区：
文献类型：
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作者：
Di Stasio, Enrico;Lancellotti, Stefano;De Cristofaro, Raimondo
具有血小板反应蛋白I型重复序列的锌蛋白酶a解整合素样和金属蛋白酶（ADAMTS 13）切割血管性血友病因子（VWF）的Tyr（1605）-Met（1606）肽键，避免超大VWF多聚体的积累。在15 ℃至45 ℃和pH值为4.5至10.5的条件下，通过荧光猝灭法（FRTS法）研究了ADAMTS 13对VWF类似物（Asp（1596）-Arg（1668）肽，荧光能量转移底物[FRTS]-VWF 73）的水解。催化作用受两个可电离基团的影响，其pK（a）值等于6.41 +/- 0.08（电离焓= 32.6 +/- 1.7 kJ/mol）和4 +/- 0.1（电离焓= 3.8 +/-0.4kJ/mol），而在Co2+取代的ADAMTS 13中，这些值分别等于6 +/-0.1和4.1 +/-0.1。FRETS-VWF 73水解的催化过程显示负活化熵（-144kJ/mol），表明过渡态比反应物的基态变得更加有序。k（cat）/K-m值与温度不呈线性相关，反映了底物的动力学“粘性”的变化。Met（1606）-Arg（1668）肽产物作为FRETS-VWF 73水解的双曲线混合型抑制剂。Asp（1653）、Glu（1655）、Glu（1660）、Asp（1663）与Thr（1656）的亲水性侧链一起显示在VWF A2序列中形成“热点”，其驱动与ADAMTS 13结合的分子识别和变构调节。VWF的Met（1606）-Arg（1668）区与ADAMTS 13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-VWF 73的分子模型表明，只有当ADAMTS 13呈现C样形状并且与VWF的酸性1653-1668区域相互作用时，底物才能适合活性位点，从而使Tyr（1605）-Met（1606）肽键正确定向，以便被活性位点中的锌-水复合物切割。
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 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 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.