Conformational Changes in Orotidine 5鈥?Monophosphate Decarboxylase: A Structure-Based Explanation for How the 5鈥?Phosphate Group Activates the Enzyme

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• 作者：Bijoy J. Desai ; B. McKay Wood ; Alexander A. Fedorov ; Elena V. Fedorov ; Bogdana Goryanova ; Tina L. Amyes ; John P. Richard ; Steven C. Almo ; John A. Gerlt
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：October 30, 2012
• 年：2012
• 卷：51
• 期：43
• 页码：8665-8678
• 全文大小：689K
• 年卷期：v.51,no.43(October 30, 2012)
• ISSN：1520-4995

文摘

The binding of a ligand to orotidine 5鈥?monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (E_o) to a closed, active conformation (E_c). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E_c路S complex) are enforced. Focusing on the OMPDC from Methanothermobacter thermautotrophicus, we find the 鈥渞emote鈥?5鈥?phosphate group of the substrate activates the enzyme 2.4 脳 10⁸-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5鈥?phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the 鈥渕obile鈥?and 鈥渇ixed鈥?half-barrel domains of the (尾/伪)₈-barrel structure. Our data support a model in which the IBE provided by the 5鈥?phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the E_c路S and E_c路S complexes relative to the E_o路S complex. The conclusion that the IBE of the 5鈥?phosphate group provides stabilization to both the E_c路S and E_c路S complexes, not just the E_c路S complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions.