Defense Date

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Medicinal Chemistry

First Advisor

Martin Safo

Abstract

Pyridoxal 5'-phosphate (PLP) is the cofactor for over 140 vitamin B6 (PLP)-dependent enzymes that are involved in various metabolic and biosynthetic pathways. Pyridoxal kinase (PL kinase) and pyridoxine 5’-phosphate oxidase (PNP oxidase) are the two key enzymes that metabolize nutritional forms of vitamin B6, including pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM) to the active cofactor form, PLP. Disruption of the PLP metabolic pathway due to mutations in PNP oxidase or PL kinase result in PLP deficiency, which is implicated in several neurological pathologies. Several ingested compounds are also known to result in PLP deficiency with concomitant neurotoxic effects. How these mutations and compounds affect B6 metabolism is not clearly understood. On the other hand, an emerging health problem is the intake of too much vitamin B6 as high doses of the reactive PLP in the cell exhibits toxic effects, including sensory and motor neuropathies. The overall aim of this research is to understand the catalytic function of PL kinase and the regulatory pathway of PLP metabolism. Using site-directed mutagenesis (Asp235Asn, Asp235Ala), kinetic and structural studies, we have shown that Asp235 may play a catalytic role in PL kinase phosphorylation activity. We also show that human PL kinase binds its substrates, PL and MgATP synergistically, and that the enzyme requires Na+ (or K+) and Mg2+ for its activity. Using kinetic study, we show severe induced MgATP substrate inhibition of PL kinase in the presence of its product, PLP, and we postulate this to be due to the formation of a non-productive ternary complex (Enzyme•PLP•MgATP). Consistently, our crystal structure of human PL kinase (2.1 Å) co-crystallized with MgATP and PLP showed both MgATP and PLP trapped at the active site. Our hypothesis is that this abortive ternary complex might be a physiological process, and that PL kinase uses this mechanism to self-regulate its activity. Our inhibition studies show theophylline, a bronchodilator as a mixed competitive inhibitor of human PL kinase with Ki of 71 μM. Our structural study (2.1 Å) shows theophylline bound at the substrate, PL binding site of human PL kinase. We also identified several potential PL kinase inhibitors from the DrugBank Chemical Compound database. Some of these compounds, including enprofylline, theobromine, caffeine, and lamotrigine, which incidentally exhibit similar neurotoxic effects as theophylline, show significant inhibitory effect on human PL kinase. Further studies are also planned to investigate the effect of these drugs on vitamin B6 metabolism in vivo.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

December 2009

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