title: A new general method for simultaneous fitting of temperature and concentration dependence of reaction rates yields kinetic and thermodynamic parameters for HIV reverse transcriptase specificity.
abstract: Recent studies have demonstrated the dominant role of induced fit in enzyme specificity of HIV reverse transcriptase and many other enzymes. However, relevant thermodynamic parameters are lacking, and equilibrium thermodynamic methods are of no avail because the key parameters can only be determined by kinetic measurement. By modifying KinTek Explorer software, we present a new general method for globally fitting data collected over a range of substrate concentrations and temperatures and apply it to HIV reverse transcriptase. Fluorescence stopped-flow methods were used to record the kinetics of enzyme conformational changes that monitor nucleotide binding and incorporation. The nucleotide concentration dependence was measured at temperatures ranging from 5 to 37 °C, and the raw data were fit globally to derive a single set of rate constants at 37 °C and a set of activation enthalpy terms to account for the kinetics at all other temperatures. This comprehensive analysis afforded thermodynamic parameters for nucleotide binding (Kd, ΔG, ΔH, and ΔSat 37 °C) and kinetic parameters for enzyme conformational changes and chemistry (rate constants and activation enthalpy). Comparisons between wild-type enzyme and a mutant resistant to nucleoside analogs used to treat HIV infections reveal that the ground state binding is weaker and the activation enthalpy for the conformational change step is significantly larger for the mutant. Further studies to explore the structural underpinnings of the observed thermodynamics and kinetics of the conformational change step may help to design better analogs to treat HIV infections and other diseases. Our new method is generally applicable to enzyme and chemical kinetics.
authors.affiliation: From the Institute for Cell and Molecular Biology, Molecular Biosciences Department, University of Texas at Austin, Austin, Texas 78712.
authors.forename: An
authors.initials: A
authors.lastname: Li
authors.affiliation: From the Institute for Cell and Molecular Biology, Molecular Biosciences Department, University of Texas at Austin, Austin, Texas 78712.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Ziehr
authors.affiliation: From the Institute for Cell and Molecular Biology, Molecular Biosciences Department, University of Texas at Austin, Austin, Texas 78712 kajohnson@mail.utexas.edu.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Johnson
mesh_terms: D003198:Computer Simulation; D003374:Coumarins / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015658:HIV Infections / Q000188:drug therapy; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D012984:Software; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013942:Thymine Nucleotides / Q000494:pharmacology*; D015215:Zidovudine / Q000494:pharmacology*
chemical_list: D003374:Coumarins; D007477:Ions; D009711:Nucleotides; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; D015215:Zidovudine; D004247:DNA; D054303:HIV Reverse Transcriptase; D008274:Magnesium; C024157:thymidine 5'-triphosphate
keywords: computer simulation; enzyme kinetics; global data fitting; human immunodeficiency virus (HIV); pre-steady-state kinetics; reverse transcription; thermodynamics