Orotidine 5'-Monophosphate Decarboxylase: Probing the Limits of the Possible for Enzyme Catalysis.
Orotidine 5'-Monophosphate Decarboxylase: Probing the Limits of the Possible for Enzyme Catalysis.
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DOI：
10.1021/acs.accounts.8b00059
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发表时间：
2018-04-17
影响因子：
18.3
通讯作者：
Reyes AC
中科院分区：
文献类型：
--
作者：
Richard JP;Amyes TL;Reyes AC
曾经被视为蛋白质黑匣子的催化作用的神秘性，随着酶-底物复合物三维结构的 X 射线晶体学测定而消失。该报道称，乳清苷5'-单磷酸脱羧酶（OMPDC）的几种高分辨率X射线晶体结构未能提供OMP酶催化脱羧形成尿苷5'-单磷酸的一致机制，因此引发了一系列争议。这种酶的 1023 倍的巨大速率加速加剧了这场争议，这“动摇了许多生物化学家对酶催化潜力的假设”。我们对 OMPDC 作用机制的研究提供了强有力的证据，表明这种酶的催化作用与效率较低的催化剂没有根本区别，同时强调了实现峰值性能水平的重要结构元素。许多酶会经历底物诱导的蛋白质构象变化，将其底物捕获在溶剂封闭的蛋白笼中，但配体与 OMPDC 结合诱导的构象变化非常复杂，这是与 OMP 的磷酸二价阴离子和核糖基底物片段形成 22 kcal/mol 的稳定结合相互作用所需的。这些片段的结合能被用来激活 OMPDC，通过从松散、开放、无配体形式的 OMPDC 创建紧密的、具有催化活性的蛋白笼，催化 OMP 乳清酸片段的脱羧。这种利用结合能进行配体驱动的构象变化提供了获得过渡态结合特异性的通用机制。碳酸底物与酶的结合所导致的速率提高部分是由于与配体结合相关的碳酸pKa的降低。 UMP 与 OMPDC 的结合导致 C-6 底物氢提取的 pKa = 29 异常大幅降低 >12 个单位，这是由于酶结合的乙烯基碳负离子（也是 OMPDC 催化脱羧的中间体）的稳定化所致。与水溶液相比，蛋白质-配体相互作用可稳定酶活性位点处的乙烯基碳负离子，而不是稳定过渡态，以通过 H+ 协调亲电子置换 CO2，从而避免形成该反应中间体。有证据表明 OMPDC 会诱导应变进入结合底物。来自磷酸二价阴离子夹环的 Gln-215 的酰胺侧链与来自 ScOMPDC 嘧啶伞的 Ser-154 的羟亚甲基侧链之间的相互作用将酰胺侧链定位为与 OMP 的磷酸二价阴离子相互作用。二阴离子夹蛋白侧链 Gln-215、Tyr-217 和 Arg-235 与脱羧过渡态的嘧啶环之间不存在直接的稳定相互作用。相反，这些侧链的作用仅仅是将 OMPDC 保持在催化活性的闭合构象中。位于离开 CO2 产物区域的 OMPDC 活性位点的疏水侧链可能通过提供该产物的疏水溶剂化作用来稳定脱羧过渡态。
The mystery associated with catalysis by what were once regarded as protein black boxes, diminished with the X-ray crystallographic determination of the three-dimensional structures of enzyme–substrate complexes. The report that several high-resolution X-ray crystal structures of orotidine 5′-monophosphate decarboxylase (OMPDC) failed to provide a consensus mechanism for enzyme-catalyzed decarboxylation of OMP to form uridine 5′-monophosphate, therefore, provoked a flurry of controversy. This controversy was fueled by the enormous 1023-fold rate acceleration for this enzyme, which had “jolted many biochemists’ assumptions about the catalytic potential of enzymes.” Our studies on the mechanism of action of OMPDC provide strong evidence that catalysis by this enzyme is not fundamentally different from less proficient catalysts, while highlighting important architectural elements that enable a peak level of performance. Many enzymes undergo substrate-induced protein conformational changes that trap their substrates in solvent occluded protein cages, but the conformational change induced by ligand binding to OMPDC is incredibly complex, as required to enable the development of 22 kcal/mol of stabilizing binding interactions with the phosphodianion and ribosyl substrate fragments of OMP. The binding energy from these fragments is utilized to activate OMPDC for catalysis of decarboxylation at the orotate fragment of OMP, through the creation of a tight, catalytically active, protein cage from the floppy, open, unliganded form of OMPDC. Such utilization of binding energy for ligand-driven conformational changes provides a general mechanism to obtain specificity in transition state binding. The rate enhancement that results from the binding of carbon acid substrates to enzymes is partly due to a reduction in the carbon acid pKa that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the pKa = 29 for abstraction of the C-6 substrate hydrogen, due to stabilization of an enzyme-bound vinyl carbanion, which is also an intermediate of OMPDC-catalyzed decarboxylation. The protein–ligand interactions operate to stabilize the vinyl carbanion at the enzyme active site compared to aqueous solution, rather than to stabilize the transition state for the concerted electrophilic displacement of CO2 by H+ that avoids formation of this reaction intermediate. There is evidence that OMPDC induces strain into the bound substrate. The interaction between the amide side chain of Gln-215 from the phosphodianion gripper loop and the hydroxymethylene side chain of Ser-154 from the pyrimidine umbrella of ScOMPDC position the amide side chain to interact with the phosphodianion of OMP. There are no direct stabilizing interactions between dianion gripper protein side chains Gln-215, Tyr-217, and Arg-235 and the pyrimidine ring at the decarboxylation transition state. Rather these side chains function solely to hold OMPDC in the catalytically active closed conformation. The hydrophobic side chains that line the active site of OMPDC in the region of the departing CO2 product may function to stabilize the decarboxylation transition state by providing hydrophobic solvation of this product.
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影响因子：
15
作者：
Fujihashi M;Ishida T;Kuroda S;Kotra LP;Pai EF;Miki K
通讯作者：
Miki K
酶体系结构：酶激活5'-单磷酸酯脱羧酶的酶激活磷酸二苯二醇相互作用的解构。
影响因子：
15
作者：
Goldman, Lawrence M.;Amyes, Tina L.;Goryanova, Bogdana;Gerlt, John A.;Richard, John P.
通讯作者：
Richard, John P.
影响因子：
2.9
作者：
Go, Maybelle K.;Amyes, Tina L.;Richard, John P.
通讯作者：
Richard, John P.
影响因子：
2.9
作者：
Goryanova B;Spong K;Amyes TL;Richard JP
通讯作者：
Richard JP
影响因子：
2.9
作者：
Goryanova B;Goldman LM;Ming S;Amyes TL;Gerlt JA;Richard JP
通讯作者：
Richard JP