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Abstract:
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We have engineered a mutant of HIV Reverse Transcriptase that can be fluorescently labeled by covalent attachment of the environmentally sensitive fluorophore 7 -diethylamino -3 - ( ( ( (2 -maleimidyl )ethyl )amino )carbonyl )coumarin (MDCC ) . The result is a polymerase that is kinetically indistinguishable from the wild -type enzyme , but provides a signal to monitor changes in enzyme structure that result from conformational changes induced by substrate binding . Using this system , we have expanded the kinetic model governing nucleotide binding to include an enzymatic isomerization following initial nucleotide binding . In doing so , we define the role of induced -fit in nucleotide specificity and mismatch discrimination . Additionally , we have characterized the kinetics governing the specificity and discrimination of several widely administered Nucleotide Reverse Transcriptase Inhibitors (NRTI’s ) used to combat HIV infection including 3TC (Lamivudine ) , FTC (Emtricitabine ) , and AZT (Zidovudine ) for the wild -type polymerase and mutants with clinical resistance to these compounds . Our findings resolve the apparent tighter binding of these inhibitor compounds compared to the correct nucleotide by showing that the affinity for the correct nucleotide is stronger than the inhibitors . The apparent weaker binding of the correct nucleotide is a result of a incomplete interpretation of binding data that fails to account for the importance of the reverse rate of the conformational change . The apparent Kd (Kd ,app ) measurements for correct nucleotide estimates Km rather than Kd because nucleotide binding does not reach equilibrium . The conformationally sensitive enzyme has also been used to characterize the kinetics governing DNA association . We show that DNA binding is governed by a two -step process where a fast initial association is followed by a second , slow isomerization that is off the pathway for nucleotide binding and incorporation . Finally , we have implemented single molecule techniques using fluorophore labeled nucleotides to study the effects of AZT incorporation on the DNA translocation dynamics of the polymerase . We find that primer termination with AZT results in DNA that fails to translocate , therefore occluding the next nucleotide from binding . This shift in translocation equilibrium exposes the newly formed phosphodiester bond to ATP - or pyrophosphate -mediated AZT excision ; thereby rescuing productive polymerization . This finding represents the first kinetic measurement of DNA translocation by a polymerase . |