Recombinant human immunodeficiency virus type 1 reverse transcriptase is heterogeneous.
Recombinant wild type (wt) and T215Y HIV-1 reverse transcriptase (RT) were isolated using three methods designated A, B, and C. The three samples of wt RT were kinetically indistinguishable with respect to dTTP turnover on poly(rA).p(dT)10. However, whereas the kinetic constants for dTTP and AZTTP for both T215Y B and T215Y C were similar to those of wt protein, T215Y A exhibited a twofold increase in Km value for dTTP and a 13-fold increase in Ki value for AZTTP with respect to wt protein purified in the same manner. We further investigated this observation by studying the denaturation of wt RT by urea. The urea denaturation curves monitored by fluorescence and circular dichroism spectroscopy were not coincident with the denaturation curve monitored by enzyme activity and yielded Cm values (the concentration of urea at which 50% of the protein is denatured) of 4.1 and 2.0 M urea, respectively. The noncoincidence of the transition curves reflects two separable, sequential, noncooperative conformational changes in the molecule: (a) from a catalytically active to an inactive conformation, and (b) from a catalytically inactive to a denatured, unfolded conformation. We therefore used denaturation as detected by changes in enzyme activity to compare the conformational stability of the three samples of wt and T215Y RT A, B, and C. The Cm values for T215Y RT did not differ from those of the respective wt; however, differences in Cm values were noted depending on how the protein was isolated. This suggested that the heterogeneity of the recombinant RT was due to small differences in conformation at or near the active site.
Division of Biochemistry, Burroughs Wellcome Co., Research Triangle Park, North Carolina, USA.
J E
JE
Wilson
L L
LL
Wright
J L
JL
Martin
S E
SE
Haire
P H
PH
Ray
G R
GR
Painter
P A
PA
Furman
D000998:Antiviral Agents / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D054306:Dideoxynucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013942:Thymine Nucleotides / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000494:pharmacology
D000998:Antiviral Agents; D054306:Dideoxynucleotides; D011993:Recombinant Fusion Proteins; D013942:Thymine Nucleotides; D015215:Zidovudine; C052873:zidovudine triphosphate; D014508:Urea; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate