An Evaluation of the Ability of Lipoamide Dehydrogenase to Catalyze the Reduction of Azides to Amines
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Authors
Collins, J. Stewart
Issue Date
1995
Type
Thesis
Language
Keywords
Alternative Title
Abstract
An azide containing compound, 3'-azido-3'-dieoxythymidine (AZT), is the
drug of choice for the treatment of HIV, the causative agent of AIDS. AZT
functions by inhibiting HIV reverse transcriptase and has demonstrated its
clinical benefits by delaying the onset of AIDS, decreasing the number of
opportunistic infections, preventing HIV infection of the fetus in pregnant
women, and effecting a partial improvement in AIDS-related dementia.
However, the benefits of AZT as a therapeutic agent are limited by its toxic
effects. At this point the mechanism and metabolites responsible for AZT induced
toxicity are relatively unknown. AZT is metabolized primarily to its 5-
O-glucuronide, but significant quantities of S'-amino-S'-deoxyfoymidine (AMT)
resulting from reduction of the 3'-azido substituent have been detected in plasma
and urine. This indicates that AMT may make a significant contribution to the
cytotoxic effects accompanying AZT therapy.
One possible mechanism of AZT bioreduction is by reactions with thiols,
which are found in all living cells. Reduction of aryl azides and AZT to the corresponding amines has been
demonstrated at physiological pH and temperature by dithiothreitol, which is
not naturally found in biological systems. However, there are numerous sources
of endogenous thiols, including the active site of the enzyme, lipoamide
dehydrogenase. This enzyme contains two subunits with a redox active disulfide
and an FAD molecule at each active site. Knowledge of the enzymes responsible
for the reduction of AZT may help improve treatment of HIV patients by
coadministering drugs that inhibit AMT formation.
The reactions of several different azides, including AZT, with lipoamide
dehydrogenase was examined in this project. The reactions were initiated with
the addition of either NADH or dihdrolipoamide, which provide the electrons
necessary for reduction. Over the course of the reaction, enzyme activity was
monitored spectrophotometrically by observing the rate of oxidation of NADH, a
substrate for the enzyme. Furthermore, the presence of reaction products was
determined by HPLC analysis. Given the data obtained in these experiments, it
can be concluded with reasonable certainty that lipoamide dehydrogenase is not
responsible for the conversion of azides to amines. Finally, inactivation of the
enzyme was observed for reactions containing NADH and the azide, which could not be effectively explained.
Description
48 p.
Citation
Publisher
Kalamazoo College
License
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