Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.
Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.
复制标题
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。
DOI：
10.1021/ja505037v
复制
发表时间：
2014-07-16
影响因子：
15
通讯作者：
Richard, John P.
中科院分区：
文献类型：
--
作者：
Goldman, Lawrence M.;Amyes, Tina L.;Goryanova, Bogdana;Gerlt, John A.;Richard, John P.
通过测定单、双和三 Q215A、Y217F 和 R235A 突变的所有组合的动力学参数 kcat 和 Km，探讨了通过抓手环和邻近该环的 R235 处的 Gln215 和 Try217 侧链与 OMP 磷酸二价阴离子相互作用来激活乳清苷 5'-单磷酸脱羧酶 (OMPDC) 的机制。磷酸二价阴离子的 12 kcal/mol 固有结合能等于 R235 (6 kcal/mol)、Q215 (2 kcal/mol)、Y217 (2 kcal/mol) 侧链以及与 G234 和 R235 主链酰胺的氢键结合能 (2 kcal/mol) 的总和。对三重突变体立方体的分析显示，磷酸二价阴离子抓手侧链之间存在较小的相互作用（约 1 kcal/mol），这与野生型 OMPDC 磷酸二价阴离子周围侧链的空间拥挤一致。这些突变导致整个底物 OMP 和底物片段 (1-β-d-赤呋喃糖基)乳清酸 (EO) 和亚磷酸二价阴离子的 OMPDC 催化反应的激活屏障发生相同的变化。这表明这些反应的过渡态通过与蛋白质催化剂的类似相互作用而稳定。 OMP 的 12 kcal/mol 固有磷酸二价阴离子结合能分为 8 kcal/mol 的结合能和 4 kcal/mol 的结合能，其中 8 kcal/mol 的结合能用于驱动游离酶热力学上不利的构象变化，导致 OMPDC 催化的 OMP 脱羧作用的 (kcat)obs 增加，而 4 kcal/mol 的结合能用于稳定米氏复合物，导致 (Km)obs 减少。
The mechanism for activation of orotidine 5′-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters kcat and Km for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-d-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (kcat)obs for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (Km)obs.
登录
查看更多内容
DOI：
10.1073/pnas.0911168107
发表时间：
2010-02-02
影响因子：
11.1
作者：
Kraut, Daniel A.;Sigala, Paul A.;Herschlag, Daniel
通讯作者：
Herschlag, Daniel
DOI：
10.1073/pnas.82.23.7840
发表时间：
1985-01-01
影响因子：
11.1
作者：
LEATHERBARROW, RJ;FERSHT, AR;WINTER, G
通讯作者：
WINTER, G
影响因子：
15
作者：
Malabanan, M. Merced;Koudelka, Astrid P.;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