Synergistic interactions of multiple mutations on catalysis during the hydroxylation reaction of p-hydroxybenzoate hydroxylase: studies of the Lys297Met, Asn300Asp, and Tyr385Phe mutants reconstituted with 8-Cl-flavin.
Synergistic interactions of multiple mutations on catalysis during the hydroxylation reaction of p-hydroxybenzoate hydroxylase: studies of the Lys297Met, Asn300Asp, and Tyr385Phe mutants reconstituted with 8-Cl-flavin.
复制标题
对羟基苯甲酸羟化酶羟化反应过程中多个突变对催化的协同相互作用：用 8-Cl-黄素重构的 Lys297Met、Asn300Asp 和 Tyr385Phe 突变体的研究。
DOI：
10.1021/bi010892v
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Massey,V
中科院分区：
文献类型：
--
作者：
Ortiz-Maldonado,M;Aeschliman,SM;Ballou,DP;Massey,V
已显示氧转移对羟基苯甲酸酯催化的对羟基苯甲酸酯羟化酶（PHBH）经由黄素的C4a-氢过氧化物发生。在促进氧从C4a-氢过氧化物向底物的转移中，两个因素可能是重要的。(a)活性位点的正静电势部分地稳定了在过渡态期间以黄素-C4a-醇盐离去基团的氧为中心的负电荷[Ortiz-Maldonado，M.，巴卢，D. P.，和Massey，V.（1999）Biochemistry 38，8124 - 8137]。(b)氢键网络电离底物以促进其对亲电C4 α-氢过氧化物中间体的亲核攻击[Entsch，B.，苍白，B。一、D.C.，D. P.，和Massey，V.（1991）J.Biol.Chem.266，17341 - 17349]。该电离也由活性位点的正静电势辅助[Moran，G. R.，Entsch，B.，苍白，B。一、和D.D. P.（1997）Biochemistry36，7548 - 7556]。黄素上的取代基可以特异性地影响醇盐离去基团的稳定性，而特定酶残基的变化可以影响活性位点和氢键网络中的电荷。我们已经使用野生型（WT）PHBH和几种突变形式，都与正常的FAD和8-Cl-FAD取代FAD，以评估两种效果的相对贡献。Lys297Met和Asn300Asp在活性位点具有减少的正电荷，并且这些变体产生比WT酶慢35倍的羟基化速率。在这些突变体形式中取代8-Cl-FAD使羟基化速率增加1.8倍，而WT用这种黄素增加≥ 4.8倍。由Tyr385Phe催化的羟基化，具有破坏的氢键网络的突变酶形式，其损害底物的电离而不改变活性位点的正电荷，通过用8-Cl-FAD取代酶而刺激1.5倍。底物，对羟基苯甲酸酯，在WT PHBH中完全电离，但由于氟取代基的吸电子效应，这种酚盐是一种差的亲核试剂。以β-对羟基苯甲酸酯为底物，在WT酶中用8-Cl-FAD取代FAD稳定了离开的醇盐，并导致与用FAD相比羟基化速率增加2.3倍。使用不与质子网络通信的底物或干扰这种相互作用的氨基酸残基的突变可以防止必要的构象变化，该构象变化允许在羟基化反应期间反应物之间的适当取向或允许最初形成的新生黄素-C4a-过氧化物阴离子的必要质子化。因此，质子网络的底物的活化和离开醇盐的稳定似乎是重要的PHBH催化的氧转移。取代基对黄素的全部作用（4.8倍）只有在最佳过渡态可以实现时才能实现，而这种最佳状态在突变体形式中不能完全实现。
The oxygen transfer top-hydroxybenzoate catalyzed byp-hydroxybenzoate hydroxylase (PHBH) has been shown to occur via a C4a-hydroperoxide of the flavin. Two factors are likely to be important in facilitating the transfer of oxygen from the C4a-hydroperoxide to the substrate. (a) The positive electrostatic potential of the active site partially stabilizes the negative charge centered on the oxygen of the flavin-C4a-alkoxide leaving group during the transition state [Ortiz-Maldonado, M., Ballou, D. P., and Massey, V. (1999)Biochemistry38, 8124−8137]. (b) The hydrogen-bonding network ionizes the substrate to promote its nucleophilic attack on the electrophilic C4a-hydroperoxide intermediate [Entsch, B., Palfey, B. A., Ballou, D. P., and Massey, V. (1991)J. Biol. Chem.266, 17341−17349]. This ionization is also aided by the positive electrostatic potential of the active site [Moran, G. R., Entsch, B., Palfey, B. A., and Ballou, D. P. (1997)Biochemistry36, 7548−7556]. Substituents on the flavin can specifically affect the stability of the alkoxide leaving-group, whereas changes to specific enzyme residues can affect the charge in the active site and the hydrogen-bonding network. We have used wild-type (WT) PHBH and several mutant forms, all with normal FAD and with 8-Cl-FAD substituted for FAD, to assess the relative contributions of the two effects. Lys297Met and Asn300Asp have decreased positive charge in the active site, and these variants engender ∼35-fold slower hydroxylation rates than the WT enzyme. Substitution of 8-Cl-FAD in these mutant forms gives ∼1.8-fold increases in hydroxylation rates, compared with a ≥4.8-fold increase for WT with this flavin. The hydroxylation catalyzed by Tyr385Phe, a mutant enzyme form with a disrupted hydrogen-bonding network that compromises the ionization of the substrate without changing the positive charge of the active site, is stimulated 1.5-fold by substituting the enzyme with 8-Cl-FAD. The substrate, tetrafluoro-p-hydroxybenzoate, is fully ionized in WT PHBH, but this phenolate is a poor nucleophile because of the electron-withdrawing effects of the fluorine substituents. With tetrafluoro-p-hydroxybenzoate as the substrate, substitution of FAD with 8-Cl-FAD in the WT enzyme stabilizes the leaving alkoxide and leads to a 2.3-fold increase in the hydroxylation rate compared to that with FAD. Either the use of substrates that do not communicate with the proton network or the mutation of amino acid residues that perturb this interaction may prevent a necessary conformational change that allows proper orientation between reactants during the hydroxylation reaction or permits the essential protonation of the initially formed nascent flavin-C4a-peroxide anion. Thus, both activation of substrate by the proton network and stabilization of the leaving alkoxide appear to be important for oxygen transfer catalyzed by PHBH. The full effect of the substituents on the flavin (4.8-fold) can only be realized when the optimal transition state can be achieved, and this optimal state is not fully realized with the mutant forms.