Investigation of substrate activation by 4-chlorobenzoyl-coenzyme A dehalogenase.
Investigation of substrate activation by 4-chlorobenzoyl-coenzyme A dehalogenase.
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4-氯苯甲酰辅酶 A 脱卤酶的底物活化研究。
DOI：
10.1021/bi962765i
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Dunaway-Mariano,D
中科院分区：
文献类型：
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作者：
Taylor,KL;Xiang,H;Liu,RQ;Yang,G;Dunaway-Mariano,D
4-氯苯甲酰辅酶A(4-CBA-CoA)脱卤酶催化4-CBA-CoA水解为4-羟基苯甲酰辅酶A(4-HBA-CoA)，利用天冬氨酸145的羧基侧链取代苯甲酰环上的C(4)上的氯。先前对酶结合底物类似物或产物配体的紫外可见、拉曼和13c核磁共振研究表明，酶活性中心的环境诱导了苯甲酰环π-电子的显著重组。这一观察结果被解释为亲电催化的证据。[4][Taylor，K.L.，Liu，R.-Q.，梁，P.-H.，Price，J.，Dunaway-Mariano，D.，Tonge，P.J.，Clarkson，J.，&Carey，P.R.(1995年)生化34,13881].脱卤酶−4-HBA-CoA复合体的最新晶体结构表明，Gly114和Phe64的主链酰胺质子参与了苯甲酰CO的两个氢键，并可能与114−121α-螺旋的正极发生偶极相互作用。与苯甲酰环紧密相连的残基包括W137、D145、W89、F64、F82和H90。本研究制备了突变株D145A、H90Q、W137F、W89F、W89Y、F64L、F82L和G114A，考察了氨基酸取代对底物苯甲酰环的催化和紫外可见光谱性质的影响。两个催化残基D145和H90的取代抑制了催化作用，但不抑制配体结合或诱导苯甲酰环吸收的红移。这两个残基似乎对底物苯甲酰环的结合或极化没有贡献。突变体F64L、F82L、W89F和W137F保持了较高的催化活性和诱导红移的能力。另一方面，W89Y突变体在催化和配体结合方面受到抑制，表明疏水性可能比堆积更重要，对苯甲酰环的结合/激活可能起关键作用。G114A突变体在底物结合和激活方面都被强烈抑制，表明H键和/或与114−121α-螺旋的偶极相互作用可能是至关重要的。
4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBA-CoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA), using the carboxylate side chain of aspartate 145 to displace the chloride from C(4) of the benzoyl ring. Previous UV-visible, Raman, and13C NMR studies of enzyme-bound substrate analog or product ligand indicated that the environment of the enzyme active site induces a significant reorganization of the benzoyl ring π-electrons. This observation was interpreted as evidence for electrophilic catalysis [viz. active-site-induced polarization of electron density away from the ring C(4)] [Taylor, K. L., Liu, R.-Q., Liang, P.-H., Price, J., Dunaway-Mariano, D., Tonge, P. J., Clarkson, J., & Carey, P. R. (1995)Biochemistry 34, 13881]. The recent crystal structure of the dehalogenase−4-HBA-CoA complex reveals two hydrogen bonds contributed to the benzoyl CO by the backbone amide protons of Gly114 and Phe64 and a possible dipolar interaction with the positive pole of the 114−121 α-helix. Residues closely surrounding the benzoyl ring include W137, D145, W89, F64, F82, and H90. In the present study, the mutants D145A, H90Q, W137F, W89F, W89Y, F64L, F82L, and G114A were prepared to examine the effect of amino acid substitution on catalysis and on perturbation of the UV-visible spectral properties of the substrate benzoyl ring. Substitution of the two catalytic residues D145 and H90 inhibited catalysis but not ligand binding or the induction of the red shift in the benzoyl ring absorption. These two residues do not appear to contribute to substrate benzoyl ring binding or polarization. The F64L, F82L, W89F, and W137F mutants retained substantial catalytic activity and the ability to induce the red shift. The W89Y mutant, on the other hand, is inhibited in catalysis and ligand binding, suggesting that hydrophobicity more than packing may be critical for the benzoyl ring binding/activation. The G114A mutant was shown to be strongly inhibited in both substrate binding and activation, indicating that H-bonding and/or interaction with the dipole of the 114−121 α-helix may be crucial.