Dissertations, Theses, and Capstone Projects

Date of Degree

2-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Ruel Z. B. Desamero

Committee Members

Lesley Davenport

Marilyn Gunner

Laura J. Juszczak

Richard Maggliozzo

Subject Categories

Analytical Chemistry | Biological and Chemical Physics | Organic Chemistry

Keywords

PKU, phenylketonuria, DHPR, mechanism, Raman spectroscopy, computational chemistry

Abstract

Human dihydropteridine reductase is an enzyme that transfers a hydride from NADH to reduce quinonoid 7,8-dihydropterin (qBH2) to 5,6,7,8-tetrahydropterin (BH4), which is a cofactor important in the production of neurotransmitters.DHPR deficiency causes a drastic form of the neurological genetic disease phenylketonuria (PKU) that does not benefit from a phenylalanine-free diet.From site-directed mutagenesis studies, mostly on Rat DHPR, we know that certain residues are important for cofactor binding, substrate binding, and hydride transfer; however, there are still some questions about how DHPR works, particularly, because there is not a crystal structure of the tertiary complex: What is the environment around the substrate pocket? Where does the compensatory proton go as the system accomplishes hydride transfer? Answering these questions are very important if we want to understand the mechanism of DHPR. Since the natural substrate is unstable, we used 6-amino-2,3-dimethylpyrimido[4,5-e][1,2,4]triazine-8-one (ADPT), which turned out to be an alternative substrate of DHPR. We probed the characteristics of this ligand in solution and in the DHPR-NAD+ complex by using isotope-edited Raman difference spectroscopy, density functional calculations, protonation state calculations, and docking simulations.

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