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In search of catalytic proficiency: The importance of enzyme conformational change to orotidine 5’-monophosphate decarboxylase catalysis

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Title: In search of catalytic proficiency: The importance of enzyme conformational change to orotidine 5’-monophosphate decarboxylase catalysis
Author(s): Wood, Bryant M.
Director of Research: Gerlt, John A.
Doctoral Committee Chair(s): Gerlt, John A.
Doctoral Committee Member(s): Kranz, David M.; Huang, Raven H.; van der Donk, Wilfred A.
Department / Program: Biochemistry
Discipline: Biochemistry
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): enzymology decarboxylase decarboxylation orotidine 5’-monophosphate decarboxylase (OMPDC) OMP decarboxylase site-directed mutagenesis viscosity effects catalytic proficiency
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 coordinate the movement of domains as well as stabilize loop closure when substrate binds. Additional residues at the opposite domain interface are currently being investigated. In order to determine the rate-limiting step and to gain a better picture of the energy landscape for OMPDC catalysis, I measured the dependence of the kinetic parameters for various OMPDCs on viscosity (2). This allowed me to determine to what degree chemistry was important to the measured rate because changing viscosity affects the rate of physical steps outside of the active-site while leaving unchanged the chemical steps secluded from solvent by the active-site. For kcat and kcat/KM for yeast OMPDC and kcat/KM for the archaeal M. thermautorophicus OMPDC, OMP decarboxylation was found to be only partially dependent on the rate of chemical steps in the enzyme (2). Therefore, the rate of carbon-carbon bond cleavage, which occurs ca. 2.8 x 10-16 s-1 in solution, is enhanced by OMPDC near to the rate at which substrate can diffuse into the active site. Furthermore, kcat/KM for a “faster” substrate, 5-fluoroOMP (FOMP), was found to be completely dependent on viscosity for the archaeal enzyme. This demonstrated that the rate of FOMP decarboxylation is limited by the rate of FOMP diffusing into the active site. This allowed for an explanation of the small difference in the kcat/KM for OMP and FOMP, 2-fold as opposed to 1000-fold as predicted. Also, evidence for slow conformational change upon substrate binding was gleaned from the inability for FOMP decarboxylation catalyzed by the yeast enzyme to reach complete dependence on solvent viscosity. In short, because the chemical rate is far too fast and because diffusive processes will exhibit linear dependence on viscosity, there must be a viscosity sensitive conformational change in the yeast enzyme. By applying the tools of enzymology learned in the Gerlt Laboratory and working successfully with numerous collaborators, I have furthered our understanding of the mechanism of one of Nature’s best catalysts, OMPDC. Increasingly in enzymology, the role of conformational change in enzyme catalysis has been recognized as an important one. This research has shed light on the conformational changes that take place when substrate binds OMPDC and how the two events are coordinated.
Issue Date: 2011-01-14
URI: http://hdl.handle.net/2142/18336
Rights Information: Copyright 2010 Bryant M. Wood
Date Available in IDEALS: 2011-01-14
Date Deposited: December 2
 

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