Substrate Specificity of Fluoroacetate Dehalogenase: An Insight from Crystallographic Analysis, Fluorescence Spectroscopy, and Theoretical Computations
Substrate Specificity of Fluoroacetate Dehalogenase: An Insight from Crystallographic Analysis, Fluorescence Spectroscopy, and Theoretical Computations
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DOI：
10.1002/chem.201103369
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发表时间：
2012-07-01
影响因子：
4.3
通讯作者：
Yoshizawa, Kazunari
中科院分区：
文献类型：
--
作者：
Nakayama, Tomonori;Kamachi, Takashi;Yoshizawa, Kazunari
通过结晶学分析、荧光光谱和理论计算，探讨了氟乙酸酯脱卤酶的高底物专一性。伯克霍尔德氏菌酶的Asp104Ala突变体的晶体结构在1.2埃分辨率下测定了FA1与氟乙酸酯的络合物。结合的氟乙酸酯的取向和构象与最近报道的沼泽红假单胞菌CGA009相应的酶突变体Asp110Asn的晶体结构中的不同(J.Am化学。SoC。2011年，133,7461)。伯克霍尔德氏菌野生型和Trp150Phe突变酶色氨酸残基的荧光测定与氟乙酸酯和氯乙酸酯孵育的FA1，以获得色氨酸残留物的环境信息。研究发现，氟乙酸酶和氯乙酸酶的色氨酸残基的环境不同，这可能是由于这两种底物在活性部位的结合方式不同所致。对接模拟和QM/MM优化用于预测底物的有利构象和取向。在最稳定的酶-氟乙酸酯络合物中，底物的F原子朝向Arg108。这是一种稳定但没有反应的构象，其中较小的O？C？F角不适合F-离子的SN2置换。C？F键的断裂是由底物在活性中心的构象变化到近攻击构象(NAC)引起的。第二低能构象对于NaC反应是合适的，C？O距离和O？C？F角对于SN2反应是合理的。在这种构象中，由于离开的F原子与周围的氨基酸残基之间存在三个氢键，激活能大大降低。氯乙酸酯由于C？CL键较长而不能达到反应构象；尽管C？CL键较弱，但这会导致活化能增加。
The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 angstrom resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzymefluoroacetate complex. This is a stable but unreactive conformation, in which the small O?C?F angle is not suitable for the SN2 displacement of the F- ion. The cleavage of the C?F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C?O distance and the O?C?F angle are reasonable for the SN2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C?Cl bond; this results in an increase of the activation energy despite the weaker C?Cl bond.