Taylor K L, Xiang H, Liu R Q, Yang G, Dunaway-Mariano D
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
Biochemistry. 1997 Feb 11;36(6):1349-61. doi: 10.1021/bi962765i.
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, and 13C 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 pi-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 C=O by the backbone amide protons of Gly114 and Phe64 and a possible dipolar interaction with the positive pole of the 114-121 alpha-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 alpha-helix may be crucial.
4-氯苯甲酰辅酶A（4-CBA-CoA）脱卤酶催化4-CBA-CoA水解生成4-羟基苯甲酰辅酶A（4-HBA-CoA），利用天冬氨酸145的羧基侧链将苯甲酰环C(4)上的氯取代。先前对酶结合的底物类似物或产物配体进行的紫外可见光谱、拉曼光谱和13C核磁共振研究表明，酶活性位点的环境会引起苯甲酰环π电子的显著重新排列。这一观察结果被解释为亲电催化的证据[即活性位点诱导电子密度从环C(4)极化][泰勒，K.L.，刘，R.-Q.，梁，P.-H.，普赖斯，J.，邓纳韦-马里亚诺，D.，汤奇，P.J.，克拉克森，J.，&凯里，P.R.（1995）《生物化学》34，13881]。脱卤酶-4-HBA-CoA复合物最近的晶体结构显示，甘氨酸114和苯丙氨酸64的主链酰胺质子与苯甲酰C=O形成了两个氢键，并且与114-121α-螺旋的正极可能存在偶极相互作用。紧密围绕苯甲酰环的残基包括色氨酸137、天冬氨酸145、色氨酸89、苯丙氨酸64、苯丙氨酸82和组氨酸90。在本研究中，制备了D145A、H90Q、W137F、W89F、W89Y、F64L、F82L和G114A突变体，以研究氨基酸取代对催化作用以及对底物苯甲酰环紫外可见光谱性质扰动的影响。两个催化残基天冬氨酸145和组氨酸90的取代抑制了催化作用，但不影响配体结合或苯甲酰环吸收的红移诱导。这两个残基似乎对底物苯甲酰环的结合或极化没有贡献。F64L、F82L、W89F和W137F突变体保留了相当大的催化活性以及诱导红移的能力。另一方面，W89Y突变体在催化和配体结合方面受到抑制，这表明对于苯甲酰环的结合/活化，疏水性比堆积可能更为关键。G114A突变体在底物结合和活化方面均受到强烈抑制，这表明氢键和/或与114-121α-螺旋偶极的相互作用可能至关重要。
