Molecular basis of the evolution of new protein functions: Kemp elimination and phosphoryl transfer reactions
type: CreativeWork
description: This is a series of investigations into the molecular basis of the evolution of new protein functions. The broad objective of this work was to determine exactly how a series of single amino acid mutations, typical of an evolutionary trajectory, can result in dramatic changes in catalytic activity, specificity and protein solubility. Various strategies were employed to achieve this aim, including analysis of existing literature concerning the various models and theories relating to molecular evolution, protein crystallography, extensive enzyme kinetics and thermodynamic analysis, theoretical analysis of catalytic mechanisms and computational simulation of protein dynamics. Three model systems were investigated: the de novo designed Kemp Eliminase (KE07), the metallo-beta-lactamases NDM1 and VIM2, and the N-acyl-homoserine lactonase AiiA. Based on these studies, I was able to identify three clear phenomena that are important in molecular evolution: first, preorganization of the active sites residues is essential for efficient catalysis; second, remote mutations are capable of causing quite drastic rearrangements to the active site and substrate binding site by modulating the conformational landscape of a protein; third, intramolecular epistasis, the way that mutations interact with each other and the sequence background that they are introduced to, can constrain evolutionary trajectories and make the evolutionary potential of a protein contingent on its starting sequence. In Chapters 3-5 I focus on KE07, performing detailed kinetic analysis of hydrogenated and deuterated substrate, which revealed entropy-enthalpy compensation in the improvement in activity as well as an unusual change in the kinetic properties in the middle of the evolutionary trajectory. This is followed by comprehensive structural analysis, which reveals the enzyme has evolved to adopt a completely unexpected active site configuration via remote mutations. Finally, using computational simulations an…
abstract: This is a series of investigations into the molecular basis of the evolution of new protein functions. The broad objective of this work was to determine exactly how a series of single amino acid mutations, typical of an evolutionary trajectory, can result in dramatic changes in catalytic activity, specificity and protein solubility. Various strategies were employed to achieve this aim, including analysis of existing literature concerning the various models and theories relating to molecular evolution, protein crystallography, extensive enzyme kinetics and thermodynamic analysis, theoretical analysis of catalytic mechanisms and computational simulation of protein dynamics. Three model systems were investigated: the de novo designed Kemp Eliminase (KE07), the metallo-beta-lactamases NDM1 and VIM2, and the N-acyl-homoserine lactonase AiiA. Based on these studies, I was able to identify three clear phenomena that are important in molecular evolution: first, preorganization of the active sites residues is essential for efficient catalysis; second, remote mutations are capable of causing quite drastic rearrangements to the active site and substrate binding site by modulating the conformational landscape of a protein; third, intramolecular epistasis, the way that mutations interact with each other and the sequence background that they are introduced to, can constrain evolutionary trajectories and make the evolutionary potential of a protein contingent on its starting sequence. In Chapters 3-5 I focus on KE07, performing detailed kinetic analysis of hydrogenated and deuterated substrate, which revealed entropy-enthalpy compensation in the improvement in activity as well as an unusual change in the kinetic properties in the middle of the evolutionary trajectory. This is followed by comprehensive structural analysis, which reveals the enzyme has evolved to adopt a completely unexpected active site configuration via remote mutations. Finally, using computational simulations an…
author: Hong, Nan-Sook
language: en
keywords: Kemp eliminase, Phosphoryl transfer, Kinetic isotope effect, enzyme evolution, protein structure and function
date published: 2016
date modified: 2026-07-24T01:07:38Z
provider: Australian Catholic University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Australian Catholic University

PropertyValue
property ID: Handle
value: http://hdl.handle.net/1885/116927