Mechanistic Dissection of Taurine Alpha-Ketoglutarate Dioxygenase (TauD): A Model Alpha-Ketoglutarate Dioxygenase
Open Access
- Author:
- Price, John C.
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 19, 2005
- Committee Members:
- Joseph M Bollinger Jr., Committee Chair/Co-Chair
Carsten Krebs, Committee Chair/Co-Chair
Squire J Booker, Committee Member
Craig Eugene Cameron, Committee Member
Michael Thomas Green, Committee Member - Keywords:
- mossbauer
TauD
enzymology
kinetics
Fe(IV)
taurine
stopped flow
freeze quench - Abstract:
- The oxidizing power of oxygen is the basis for the function of the respiratory cycle and is used in many biosynthetic processes. Harnessing these oxidizing equivalents requires multiple levels of precaution and precise control. Even so preventing and repairing the damage due to reduced oxygen species is a constant effort in aerobic organisms. The Fe(II)•alpha-ketoglutarate dioxygenase enzymes employ a mechanism which requires the decarboxylation of alpha-ketoglutarate to create a highly oxidized Fe center. These enzymes are very effective at specific two electron oxidations of unactivated carbon atoms, and occupy key positions in a surprising number of biological systems. This study describes the mechanistic dissection of a model Fe(II)•alpha-ketoglutarate dioxygenase, taurine dioxygenase (TauD). Contributions made through study of this system have answered questions postulated more than 20 years ago. Chapter 1 introduces general features of the enzyme family, and summarizes the literature describing what was known prior to this study regarding the mechanism of action for these enzymes. Chapter 1 also introduces several members of the family using these systems to describe the utility of the family and the importance of understanding their mechanism of action. Chapter 2 presents all the work done in kinetically characterizing the model system (TauD) describing formation of the reactive enzyme complex and detection of intermediates that accumulate within the catalytic cycle. Chapter 3 describes the use of sophisticated spectroscopy and a synthetic substrate isotopomer to characterize the two accumulating intermediates chemically and structurally. Chapter 4 explores the effect of substrate binding and the unproductive reaction(s) that occur in the absence of substrate. Chapter 5 contains studies from a variety of alternative substrates which offer interesting, yet not fully developed insights into the delicate balance that allows the active site to activate oxygen and successfully utilize the resulting oxidizing equivalents so effectively and specifically.