Catalysis by orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.
Catalysis by orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.
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DOI：
10.1021/bi301650d
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发表时间：
2013-01-22
期刊：
影响因子：
2.9
通讯作者：
Richard JP
中科院分区：
文献类型：
--
作者：
Goryanova B;Spong K;Amyes TL;Richard JP
报道了乳清酸苷5 ′-单磷酸脱羧酶（OMPDC）天然底物乳清酸苷5 ′-单磷酸（OMP）的两个截短类似物1-（β-D-赤呋喃糖基）-5-氟乳清酸（FEO）和5′-脱氧-5-氟乳清酸苷（5′-dFO）的合成。通过比较OMPDC催化的FEO（10 M−1 s−1）和1-（β-D-赤呋喃糖基）乳清酸（EO，0.026 M−1 s−1）的脱羧反应的二级速率常数，发现通过与FEO的5-F取代基的相互作用，乙烯基碳负离子类过渡态的稳定性为3.5 kcal/mol。OMPDC催化的FEO和EO的脱羧反应都被外源性亚磷酸根二价阴离子（HPO 32-）激活，但5-F取代基仅导致FEO的亚磷酸根激活反应的过渡态稳定0.8 kcal。这提供了强有力的证据表明，亚磷酸盐活化的OMPDC催化的FEO反应不受酶结合底物脱羧的化学步骤的限制。有证据表明，从EO的亚磷酸盐活化反应的脱羧化学步骤到磷酸盐夹持环的闭合以及FEO反应的三元E·FEO·HPO 32 −复合物处的酶构象变化，限速步骤发生了变化。5′-dFO和乳清酸苷的4′-CH 3和4′-CH 2 OH基团分别导致2.9 kcal/mol未活化脱羧的过渡态的相同去稳定化。相比之下，5′-dFO的4′-CH 3基团和乳清酸苷的4′-CH 2 OH基团导致亚磷酸盐活化脱羧的过渡态的非常不同的4.7和8.3 kcal/mol不稳定。在这里，5′-dFO上的4′-CH 3取代基的失稳作用被限速构象变化所掩盖，这种构象变化降低了母体底物FEO的亚磷酸盐活化反应的三级速率常数。
The syntheses of two novel truncated analogs of the natural substrate orotidine 5′-monophosphate (OMP) for orotidine 5′-monophosphate decarboxylase (OMPDC) with enhanced reactivity towards decarboxylation are reported: 1-(β-D-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5′-deoxy-5-fluoroorotidine (5′-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M−1 s−1) and 1-(β-D-erythrofuranosyl)orotic acid (EO, 0.026 M−1 s−1) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO32−), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E·FEO·HPO32− complex for the reaction of FEO. The 4′-CH3 and 4′-CH2OH groups of 5′-dFO and orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4′-CH3 group of 5′-dFO and the 4′-CH2OH group of orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4′-CH3 substituent at 5′-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.
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影响因子：
15
作者：
Toth, Krisztina;Amyes, Tina L.;Richard, John P.
通讯作者：
Richard, John P.
影响因子：
7.3
作者：
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通讯作者：
Kotra, Lakshmi P.
影响因子：
6.8
作者：
Malabanan, M. Merced;Amyes, Tina L.;Richard, John P.
通讯作者：
Richard, John P.
影响因子：
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作者：
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通讯作者：
JENCKS, WP
影响因子：
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作者：
RADZICKA, A;WOLFENDEN, R
通讯作者：
WOLFENDEN, R