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Wöhrl, Birgitta M. ; Krebs, Ruth ; Thrall, Sara H. ; Le Grice, Stuart F.J. ; Scheidig, Axel J. ; Goody, Roger S.:
Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip grip region of p66 : implications for DNA synthesis and dimerization.
In: The Journal of Biological Chemistry.
Bd. 272
(1997)
.
- S. 17581-17587.
ISSN 1083-351X
DOI: https://doi.org/10.1074/jbc.272.28.17581
Abstract
The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the β12-β13 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 p66W229A/51 and 23-fold for mutant p66G231A/51, and the maximal pre-steady-state rate of nucleotide incorporation,k pol, was reduced 27-fold for p66W229A/51 and 70-fold for p66G231A/51, respectively. Mutant p66M230A/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.
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| Publikationsform: | Artikel in einer Zeitschrift |
|---|---|
| Begutachteter Beitrag: | Ja |
| Institutionen der Universität: | Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Lehrstuhl Biochemie mit Schwerpunkt Biophysikalische Chemie Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Ehemalige Professoren > Lehrstuhl Biopolymere - Apl. Prof. Dr. Birgitta Wöhrl Fakultäten Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Ehemalige Professoren |
| Titel an der UBT entstanden: | Nein |
| Themengebiete aus DDC: | 500 Naturwissenschaften und Mathematik > 540 Chemie 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie |
| Eingestellt am: | 20 Mai 2019 06:43 |
| Letzte Änderung: | 20 Mai 2019 06:43 |
| URI: | https://eref.uni-bayreuth.de/id/eprint/48994 |