In search of catalytic proficiency: The importance of enzyme conformational change to orotidine 5’-monophosphate decarboxylase catalysis
type: CreativeWork
description: The focus of this research is the role of conformational flexibility in catalysis by a TIM-barrel enzyme in pyrimidine biosynthesis, orotidine 5’-monophosphate decarboxylase (OMPDC). OMPDC catalyzes the decarboxylation of OMP to UMP; the uncatalyzed rate for this reaction has been estimated to be 2.8 x 1016 s-1 (1). The slow rate without OMPDC is attributable to the lack of internal stabilization of the negative charge which must develop in the intermediate after decarboxylation. Because OMPDC does not utilize a cofactor in its mechanism, discovering how it is able to enhance this very slow rate to near the limits of diffusion is an important problem, kcat/KM = 1.3 x 107 M-1s-1 (2). Through alanine-scanning mutagenesis, I have identified important residues in OMPDC catalysis (3, 4). The large impact of mutating residues on the periphery of the active-site has helped develop an understanding of the importance of conformational change. Residues Ser127 and Gln185 from two different loops form an interaction that helps to coordinate loop closure with substrate binding; these residues also interact with the substrate (4). Besides active-site loop closure, crystal structures reveal the TIM-barrel of OMPDC to function as two halves which move toward one another when ligand binds. Near one of the boundaries between these two domains, I identified residues remote from the active-site which form a hydrophobic cluster in the “closed” state of the enzyme; Val182 from the mobile active-site loop becomes anchored in this cluster upon loop closure (3). Through site-directed mutagenesis, enzyme assays, and collaboration with X-ray crystallography experts in the Almo Group at Columbia University, I was able to determine that these hydrophobic interactions were important specifically to conformational change from an “open” to a “closed” state of the enzyme and that mutations to these residues had little impact on the “closed” state itself (3). It is thought that this cluster helps to…
abstract: The focus of this research is the role of conformational flexibility in catalysis by a TIM-barrel enzyme in pyrimidine biosynthesis, orotidine 5’-monophosphate decarboxylase (OMPDC). OMPDC catalyzes the decarboxylation of OMP to UMP; the uncatalyzed rate for this reaction has been estimated to be 2.8 x 1016 s-1 (1). The slow rate without OMPDC is attributable to the lack of internal stabilization of the negative charge which must develop in the intermediate after decarboxylation. Because OMPDC does not utilize a cofactor in its mechanism, discovering how it is able to enhance this very slow rate to near the limits of diffusion is an important problem, kcat/KM = 1.3 x 107 M-1s-1 (2). Through alanine-scanning mutagenesis, I have identified important residues in OMPDC catalysis (3, 4). The large impact of mutating residues on the periphery of the active-site has helped develop an understanding of the importance of conformational change. Residues Ser127 and Gln185 from two different loops form an interaction that helps to coordinate loop closure with substrate binding; these residues also interact with the substrate (4). Besides active-site loop closure, crystal structures reveal the TIM-barrel of OMPDC to function as two halves which move toward one another when ligand binds. Near one of the boundaries between these two domains, I identified residues remote from the active-site which form a hydrophobic cluster in the “closed” state of the enzyme; Val182 from the mobile active-site loop becomes anchored in this cluster upon loop closure (3). Through site-directed mutagenesis, enzyme assays, and collaboration with X-ray crystallography experts in the Almo Group at Columbia University, I was able to determine that these hydrophobic interactions were important specifically to conformational change from an “open” to a “closed” state of the enzyme and that mutations to these residues had little impact on the “closed” state itself (3). It is thought that this cluster helps to…
author: Wood, Bryant M.
language: en
keywords: enzymology, decarboxylase, decarboxylation, orotidine 5’-monophosphate decarboxylase (OMPDC), OMP decarboxylase, site-directed mutagenesis, viscosity effects, catalytic proficiency
date published: 2011-01-14T22:46:41Z
date modified: 2026-07-22T22:25:11Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/18336