Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66. Implications for DNA synthesis and dimerization.
The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the beta12-beta13 hairpin (residues 227-235). It has been proposed to play a role in aligning the 3'-OH end of the primer in a position for nucleophilic attack on an incoming dNTP. To analyze the importance of the primer grip for RT function, mutant RTs were used that contain single alanine substitutions of residues Trp229, Met230, Gly231, and Tyr232 in the p66 subunit of the heterodimeric p66/51 enzyme. Steady-state and pre-steady-state kinetic analyses of the enzymes were performed. All mutant enzymes revealed reduced polymerase activity. Mutation of Y232A showed the smallest effect on polymerase function. Equilibrium fluorescence titrations demonstrated that the affinity of the mutants for tRNA was only slightly affected. However, the affinity for primer-template DNA was reduced 27-fold for mutant p66(W229A)/51 and 23-fold for mutant p66(G231A)/51, and the maximal pre-steady-state rate of nucleotide incorporation, kpol, was reduced 27-fold for p66(W229A)/51 and 70-fold for p66(G231A)/51, respectively. Mutant p66(M230A)/51 revealed no reduced affinity for primer-template but showed a 71-fold reduced affinity for dTTP. Additionally, the mutations Trp229 and Gly231 affected the stability of the RT heterodimer.
Wöhrl
B M
BM
Max Planck Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Rheinlanddamm 201, 44139 Dortmund, Germany. birgit.woehrl@mpi-dortmund.mpg.de
Krebs
R
R
Thrall
S H
SH
Le Grice
S F
SF
Scheidig
A J
AJ
Goody
R S
RS
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004261:DNA Replication; D019281:Dimerization; D004795:Enzyme Stability; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D012357:RNA, Transfer, Lys / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism
D017931:DNA Primers; D012357:RNA, Transfer, Lys; D013942:Thymine Nucleotides; D004247:DNA; D054303:HIV Reverse Transcriptase; C024157:thymidine 5'-triphosphate