Reverse transcriptase is an enzyme essential for replication of the Human Immunodeficiency Virus (HIV) and the target of the therapeutic drug Zidovudine (AZT). An in-depth understanding of the enzyme mechanism and the interaction of inhibitors could ultimately lead to drugs which are less toxic and more selective and effective. The current proposal seeks to understand at a molecular level the three enzymatic activities of reverse transcriptase, the interaction of nucleoside inhibitors such as AZT and non-nucleoside inhibitors such as neviparine and TIBO as well as resistance to these compounds which develops through mutation. The current proposal is based upon a rapid transient kinetic approach using rapid chemical quench and stopped-flow fluorescence methodology. The important advantage to this approach is the ability to observe events occurring at the active site including binding events, protein conformation changes, and the catalysis. The rate constants of individual steps can be measured directly and any enzyme intermediates which might be formed can be observed directly.
The specific aims of this proposal are: 1. Provide a complete kinetic and thermodynamic description of the enzymatic reaction pathway for Reverse Transcriptase. This will include an understanding of polymerase activity with RNA and DNA substrates and RNAse cleavage activity and how these catalytic activities are coordinated with one another. 2. Examine the kinetics of misincorporation of incorrect nucleotides and develop an understanding as to how this contributes to the overall fidelity and processivity of the enzyme. 3. Examine the mechanism of inhibition of nucleoside analogs such as AZT. Identify factors important for governing interaction with the enzyme. 4. Determine the mechanism of inhibition of non-nucleoside analogs such as the benzodiazepine like compounds such as TIBO inhibitors and characterize their interaction with the enzyme. 5. Explore the basis for resistance to AZT and non-nucleoside analogs by providing a complete kinetic characterization of mutant reverse transcriptases.
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