Structural requirements for enzymatic efficiency in cofactor-independent decarboxylation
type: CreativeWork
description: Orotidine 5’-monophosphate decarboxylase (OMPDC) is the last enzyme in the de novo pyrimidine biosynthetic pathway. It catalyzes the decarboxylation of orotidine 5’-monophosphate (OMP) to uridine 5’-monophosphate (UMP), without the requirement of a cofactor. It provides catalytic rate enhancements of ~1017, compared to the uncatalyzed reaction, making it one of the most efficient enzymes known. OMPDC has thus been a paradigm to understand enzymatic catalysis. Yet, many aspects of its catalytic mechanism are not well understood. The reaction coordinate consists of a vinyl carbanion/carbene intermediate. To characterize the interaction of the intermediate with the enzyme active site, I performed site-directed mutagenesis of Ser 127 to L-glycerate residue (Chapter 2). This was accomplished by in vitro protein expression using L-glycerate charged tRNA. Examination of kinetic parameters of this mutant revealed that the backbone interactions accounts for 102 fold of the total 1017 fold rate enhancement. OMPDC achieves its extraordinary rate enhancements by ground-state destabilization (GSD) and transition state destabilization (TSS). These strategies, enforced in the Michaelis complex, are accompanied by conformational changes, as characterized by the Apo (Eo) and inhibitor bound (Ec) crystal structures. A hallmark of these conformational changes is the selective formation of interactions in the Ec form compared to Eo, by residues remote from the active site of the enzyme. The role of these conformational changes in OMPDC catalysis has not been fully understood. Using a combination of site-directed mutagenesis, substrate analogs, enzyme kinetic analysis and x-ray crystallographic analysis, research in Chapter 3 attempts to elucidate the mechanism for the formation of Ec from Eo. By comparing the second order rate constants (kcat/KM) for decarboxylation of OMP and a substrate analog devoid of 5’-phosphate group (EO) for mutants of residues that interact with the 5’-phospha…
abstract: Orotidine 5’-monophosphate decarboxylase (OMPDC) is the last enzyme in the de novo pyrimidine biosynthetic pathway. It catalyzes the decarboxylation of orotidine 5’-monophosphate (OMP) to uridine 5’-monophosphate (UMP), without the requirement of a cofactor. It provides catalytic rate enhancements of ~1017, compared to the uncatalyzed reaction, making it one of the most efficient enzymes known. OMPDC has thus been a paradigm to understand enzymatic catalysis. Yet, many aspects of its catalytic mechanism are not well understood. The reaction coordinate consists of a vinyl carbanion/carbene intermediate. To characterize the interaction of the intermediate with the enzyme active site, I performed site-directed mutagenesis of Ser 127 to L-glycerate residue (Chapter 2). This was accomplished by in vitro protein expression using L-glycerate charged tRNA. Examination of kinetic parameters of this mutant revealed that the backbone interactions accounts for 102 fold of the total 1017 fold rate enhancement. OMPDC achieves its extraordinary rate enhancements by ground-state destabilization (GSD) and transition state destabilization (TSS). These strategies, enforced in the Michaelis complex, are accompanied by conformational changes, as characterized by the Apo (Eo) and inhibitor bound (Ec) crystal structures. A hallmark of these conformational changes is the selective formation of interactions in the Ec form compared to Eo, by residues remote from the active site of the enzyme. The role of these conformational changes in OMPDC catalysis has not been fully understood. Using a combination of site-directed mutagenesis, substrate analogs, enzyme kinetic analysis and x-ray crystallographic analysis, research in Chapter 3 attempts to elucidate the mechanism for the formation of Ec from Eo. By comparing the second order rate constants (kcat/KM) for decarboxylation of OMP and a substrate analog devoid of 5’-phosphate group (EO) for mutants of residues that interact with the 5’-phospha…
author: Desai, Bijoy
language: en
keywords: Enzymology, catalysis, Orotidine 5'-monophosphate decarboxylase, in vitro translation, orthogonal translation system, non natural protein residue, conformational change, intrinsic binding energy, frsA
date published: 2015-01-21T19:49:25Z
date modified: 2026-07-22T22:26:07Z
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/2142/72911