A Caltech Library Service

Methods for Computational Enzyme Design and Application to the Chorismate-Prephenate Rearrangement


Lassila, Jonathan Kyle (2007) Methods for Computational Enzyme Design and Application to the Chorismate-Prephenate Rearrangement. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/4xm1-h147.


The Claisen rearrangement of chorismate to prephenate has become an important model system for developing understanding of enzymatic catalysis as well as for computational treatment of enzyme active sites. This thesis presents general methods for the computational design of enzyme active sites and applies these methods to the design of catalysts for the chorismate-prephenate rearrangement. The computational methods described allow the incorporation of transition-state structures and other small molecules into protein design calculations. These design procedures were tested through redesign of the active site of Escherichia coli chorismate mutase. The six predicted mutations were experimentally characterized and most maintained or increased the catalytic activity of the enzyme. To further investigate the context of the mutations predicted in the calculation and the tolerance of a natural enzyme to secondary active site mutations, extensive substitution experiments were performed. The effect of every amino acid in five active site hydrophobic positions and one N-capping position was evaluated. These experiments clarified some of the strengths and weaknesses of the computational modeling procedure. Finally, attempts to design a completely new enzyme for catalysis of the chorismate-prephenate rearrangement are discussed.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:computational library design; directed evolution; pericyclic reactions; protein-ligand interactions; sigmatropic reactions; site-saturation mutagenesis
Degree Grantor:California Institute of Technology
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Mayo, Stephen L.
Thesis Committee:
  • Bjorkman, Pamela J. (chair)
  • Rees, Douglas C.
  • Arnold, Frances Hamilton
  • Mayo, Stephen L.
Defense Date:15 November 2006
Record Number:CaltechETD:etd-12182006-150535
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5046
Deposited By: Imported from ETD-db
Deposited On:19 Dec 2006
Last Modified:15 Apr 2020 23:39

Thesis Files

PDF (lassila_thesis.pdf) - Final Version
See Usage Policy.


Repository Staff Only: item control page