Metalloprotein Engineering Using Heme Protein Scaffolds to Investigate the Oxidation of Endogenous Aromatic Amino Acids and Exogenous Substrates
Pfister, Thomas Daniel
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https://hdl.handle.net/2142/84833
Description
Title
Metalloprotein Engineering Using Heme Protein Scaffolds to Investigate the Oxidation of Endogenous Aromatic Amino Acids and Exogenous Substrates
Author(s)
Pfister, Thomas Daniel
Issue Date
2006
Doctoral Committee Chair(s)
Lu, Yi
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Inorganic
Language
eng
Abstract
Protein engineering by rational design has been used to study heme proteins and artificial Mn-salen containing metalloenzymes using heme protein scaffolds. Heme proteins perform a wide variety of functions including the catalysis of numerous different reactions. Some of the structural features involved in conferring these functional properties have been examined in this thesis. Heme protein models have been made of peroxidases and P450 to gain insight into the native function of these enzymes. The role of role of redox active amino acids in heme proteins was studied by mutating Trp an Tyr residues in cytochrome c peroxidase (CcP). Sequential mutation of these residues resulting in increased lifetime of the compound I intermediate. An Engineered Trp in the active site of myoglobin (Mb) was used to mimic bound substrate. The Trp was stoichiometrically hydroxylated to 6-hydroxytryptophan. Factors required to engineer metal binding sites were studied by the redesign of an engineered manganese binding site in CcP. Kinetic parameters and crystal structures of the protein demonstrate the importance of the metal and ligands in engineering metal binding sites. Finally the role of cofactors in catalysis was probed by dually anchoring a Mn-salen complex in the active site of a heme protein scaffold (Mb). Dual anchoring was shown to increase ee and rate in this artificial metalloenzyme. It is concluded that the control of reactive intermediates, presence of appropriate substrate binding sites and the choice of an appropriate scaffold and cofactor are essential in engineering protein models and biocatalysts.
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