UGTs are of particular physiological, pharmacological, and toxicological significance due to their involvement in biotransformation of the largest number of endogenous compounds, drugs, and environmental toxins of different chemical structures and origins of any phase II metabolizing enzyme. Moreover, some UGT substrates, which are nuclear receptor ligands, signaling molecules, important drugs, and environmental chemicals, can affect UGT mRNA expression and alter the activity of the UGT enzymes. There is a tremendous need for the understanding of the pharmacogenetics and drug interactions of chemicals that are substrates for and inducers of UGTs. Due to the lack of homogeneous proteins for structural studies, it was previously impossible to predict the molecular mechanism of glucuronidation required to understand the mechanistic aspects of UGT substrate specificity, inhibition, and drug-drug interactions. Now that homogeneous UGT proteins have been generated, studies identifying UGT active sites are proposed. Our central hypothesis is that the N-terminal domain of UGTs consists of distinct substrate recognition sequences (SRSs) that display specificity for classes of UGT substrates and, thus, determine the catalytic mechanism and substrate specificity of individual UGT isoforms. For the co- substrate active site, we hypothesize that the site of UDP/Mg2+ binding is localized in the highly conserved C-terminal end of the UGT molecule. Specifically, we postulate that a DxxD motif, conserved in all UGTs. is a major element of the UDP-GlcUA (co-substrate) binding site. These hypotheses will be tested by the following Specific Aims: 1. To identify and characterize the substrate recognition sequences of UGTs. 2. To identify and characterize the UDP-GlcUA binding site of UGTs. 3. To sequence photolabeled polypeptides by mass spectrometry. 4. To modify amino acid motifs of putative binding sites by site-directed mutagenesis and catalytically characterize the generated mutants. The information derived from the proposed studies will provide insight into the molecular mechanism of glucuronidation and will enhance our ability to predict and direct the metabolic transformation of both endogenous and exogenous substrates for UGTs. This information will be important for understanding specific interactions between endogenous substrates and drugs. It will also serve as a basis in the search for new drugs and inhibitors of specific UGT isoforms, thus affecting drug therapy, drug-drug interactions, and the risk of chemically-induced diseases, including cancer. ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM075893-05A1
Application #
7143067
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
1996-06-01
Project End
2010-07-31
Budget Start
2006-08-04
Budget End
2007-07-31
Support Year
5
Fiscal Year
2006
Total Cost
$283,977
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
122452563
City
Little Rock
State
AR
Country
United States
Zip Code
72205
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