at Google ScholarTitle data
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.
Vol. 272
(1997)
.
- pp. 17581-17587.
ISSN 1083-351X
DOI: https://doi.org/10.1074/jbc.272.28.17581
Abstract in another language
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.
Further data
| Item Type: | Article in a journal |
|---|---|
| Refereed: | Yes |
| Institutions of the University: | Faculties > Faculty of Biology, Chemistry and Earth Sciences Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry with an Emphasis on Biophysical Chemistry Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Chair Biopolymers - Apl. Prof. Dr. Birgitta Wöhrl Faculties Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors |
| Result of work at the UBT: | No |
| DDC Subjects: | 500 Science > 540 Chemistry 500 Science > 570 Life sciences, biology |
| Date Deposited: | 20 May 2019 06:43 |
| Last Modified: | 20 May 2019 06:43 |
| URI: | https://eref.uni-bayreuth.de/id/eprint/48994 |