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CHARACTERIZATION OF HIV‐1 REVERSE TRANSCRIPTASE 3TC SPECIFICITY BY CONFORMATIONALLY SENSITIVE FLUORESCENCE REVEALS NEW INSIGHTS INTO THE KINETIC BASIS OF INHIBITOR DISCRIMINATION
Author(s) -
Kellinger Matthew William,
Johnson Kenneth Allen
Publication year - 2010
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.24.1_supplement.681.10
Subject(s) - nucleotide , chemistry , dissociation constant , reverse transcriptase , stereochemistry , enzyme , reaction rate constant , crystallography , kinetics , biochemistry , rna , physics , receptor , quantum mechanics , gene
HIV‐1 Reverse Transcriptase (HIV‐RT) nucleotide specificity is best quantified by analysis of the concentration dependence of the rate using single turnover rapid Quench‐Flow methods which provide a rate of polymerization (k pol ) and an apparent dissociation constant (K d ) such that k pol /K d = k cat /K m . Analysis of nucleoside analog RT inhibitors (NRTIs) has led to the surprising conclusion that most appear to bind more tightly than normal nucleotides. Using an enzyme linked fluorophore, we show that nucleotide binding is a two step process involving weak nucleotide ground state binding, followed by a conformational change from an “open” to “closed” state. These steps together define the true K d for nucleotide binding at equilibrium. We show that contrary to previously reported findings, the dCTP analog 3TC binds 8‐fold more weakly to the enzyme than the correct nucleotide. The result is a specificity constant (k cat /K m ) of 9.7μM −1 s −1 for dCTP and 0.7μM −1 s −1 for 3TC. The specificity constant for dCTP is determined solely by the rate of nucleotide binding (k cat /K m = K 1 k 2 in the two‐step sequence), whereas the slower chemical reaction (k 3 ) for 3TC incorporation allows the binding and isomerization to reach equilibrium so that k cat /K m = k pol /K 1 K 2 . This work provides mechanistic basis for discrimination of 3TC, and corrects how K m , K d , and K d,app must be assigned for NRTIs.