Kinetics of association and dissociation of HIV-1 reverse transcriptase subunits.
The biologically active form of HIV-1 reverse transcriptase (RT) is the p66/p51 heterodimer. The process of maturation of the heterodimer from precursor proteins is poorly understood. Previous studies indicated that association of p66 and p51 is very slow. Three techniques, a pre-steady-state activity assay, intrinsic tryptophan fluorescence, and a FRET assay, were used to monitor the dimerization kinetics of RT. Kinetic experiments were conducted with purified p66 and p51 proteins in aqueous buffer. All three techniques gave essentially the same results. The dissociation kinetics of p66/p51 were first-order with rate constants (k(diss)) of approximately 4 x 10(-6) s(-1) (t(1/2) = 48 h). The association kinetics of p66 and p51 were concentration-dependent with second-order rate constants (k(ass)) of approximately 1.7 M(-1) s(-1) for the simple bimolecular association reaction. The implications of slow dimerization of p66/p51 for the maturation process are discussed. A reaction-controlled model invoking conformational selection is proposed to explain the slow protein-protein association kinetics.
Venezia
Carl F
CF
Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
Meany
Brendan J
BJ
Braz
Valerie A
VA
Barkley
Mary D
MD
D019943:Amino Acid Substitution; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D014364:Tryptophan; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase