An Evaluation of the Ability of Lipoamide Dehydrogenase to Catalyze the Reduction of Azides to Amines
Collins, J. Stewart
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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.