The Alpha-ketoacid-dependent dioxygenases are a class of enzymes which catalyze the hydroxylation of aliphatic and aromatic carbon atoms in a variety of substrates. These iron containing proteins are vital to amino acid degradation, the biosynthesis of carnitine, and the post-translational modification of collagen, but their mechanisms of catalysis are poorly understood. We will use modified substrates of these enzymes as probes of their modes of action. Specifically, we will: 1) Purify two representative examples of these proteins -- p-hydroxyphenylpyruvate dioxygenase (which catalyzes the second reaction of tyrosine catabolism) and Gamma-butyrobetaine dioxygenase (which catalyzes the last step in carnitine biosynthesis) -- by literature procedures. 2) Synthesize isotopically-labeled substrates and potential alternate substrates containing mechanistically informative reactive functionality at key sites of the molecules. 3) Incubate these compounds with the enzymes, determine their competence as substrates and/or inhibitors, and determine the structures of the incubation products in order to address the following mechanistic points of general importance to the understanding of all Alpha-ketoacid dioxygenases: a) The nature of the highly reactive carbon oxygenating agent. b) The stereochemical course of oxygenation. c) The mechanism and function of Alpha-ketoacid oxidative decarboxylation. d) The possible presence and role of radical and carbanionic intermediates in catalysis. Evaluation of the results from these experiments will aid in forming a conclusion about the nature and sequence of events at the active sites of these important enzymes during catalysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031801-05
Application #
3280121
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1983-09-01
Project End
1989-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Princeton University
Department
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
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