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Characterization of HIV‐1 Reverse Transcriptase Nucleotide Specificity by Conformationally Sensitive Fluorescence
Author(s) -
Kellinger Matthew William,
Piasecki Shawn,
Johnson Kenneth Allen
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.853.1
Subject(s) - nucleotide , reverse transcriptase , chemistry , fluorophore , kinetics , fluorescence , enzyme , mutant , enzyme kinetics , human immunodeficiency virus (hiv) , stereochemistry , biochemistry , biophysics , resistance mutation , active site , biology , rna , virology , physics , quantum mechanics , gene
Site‐specific labeling of HIV‐1 Reverse Transcriptase (HIVRT) with a conformationally sensitive fluorophore has been optimized to allow for transient kinetic characterization of steps preceding and following chemistry. The labeling did not affect the kinetics of incorporation. Pre‐steady state Quench‐flow data has been globally fit with Stopped‐flow fluorescence data to support a minimal two‐step model for nucleotide binding. Initial ground state binding is followed by a rate limiting conformational change which results in a 10‐fold decrease in the overall Kd for correct nucleotide. Kinetic partitioning of the closed complex then commits the enzyme to chemical incorporation rather than nucleotide release. This work provides the first comprehensive characterization of an induced fit model for HIVRT, and has been used to further understand mechanisms of resistance of 3TC by the M184V HIVRT mutant.