Identification of an Acyl-Enzyme Intermediate in a meta-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad
Identification of an Acyl-Enzyme Intermediate in a meta-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad
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DOI：
10.1021/ja208544g
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发表时间：
2012-03-14
影响因子：
15
通讯作者：
Eltis, Lindsay D.
中科院分区：
文献类型：
--
作者：
Ruzzini, Antonio C.;Ghosh, Subhangi;Eltis, Lindsay D.
Meta-cleavage product （MCP）水解酶是α / β -水解酶超家族的成员，利用Ser-His-Asp三联体催化C-C键的水解。BphD是来自联苯降解途径的MCP水解酶，它将2-羟基-6-氧-6-苯基六酸-2,4-二烯酸（HOPDA）水解为2-羟基五酸-2,4-二烯酸（HPD）和苯甲酸酯。HOPDA培养的BphD H265Q的1.6埃分辨率晶体结构表明，该酶的催化丝氨酸被苯甲酰化。酰基酶是由氧阴离子空穴残基的酰胺主链形成的氢键稳定的，这与Ser112亲核攻击时形成的四面体氧阴离子一致。化学猝灭和质谱研究证实了野生型BphD中ser112 -苯甲酰物种的形成和衰变，其时间尺度与(H2O)-O-18水解过程中产生的苯甲酸酯的转换和单一等量O-18的结合一致。快速扫描动力学研究表明，催化组氨酸对酰化速率的贡献仅为一个数量级，但对去酰化速率的影响超过5个数量级。之前在野生型酶中检测到的橙色催化中间体ESred，在本文中被认为是碳离子，在H265Q的水解过程中没有观察到。在新提出的机制中，碳离子从Ser112中提取了一个质子，从而完成了变异构，并产生了丝氨酸，用于对c6 -羰基的亲核攻击。最后，定量观察到的预稳态动力学爆发表明BphD是一种半位点活性酶。虽然更新的催化机制与丝氨酸蛋白酶具有相同的特征，但MCP水解酶特异性化学强调了Ser-His-Asp三联体的多功能性。
Meta-cleavage product (MCP) hydrolases are members of the alpha/beta-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 angstrom resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of O-18 into the benzoate produced during hydrolysis in (H2O)-O-18. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over S orders of magnitude. The orange-colored catalytic intermediate, ESred, previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophific attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.