Abstract:
:The majority of pre-steady-state kinetic investigations with HIV-1 reverse transcriptase (HIV-1 RT) have reported substoichiometric bursts (30-50%) of product formation in the initial reaction cycle. By using quantitative amino acid analysis, we have revised the extinction coefficient of the HIV-1 RT heterodimer and show that normal nucleotide incorporation (canonical four bases) proceeds with quantitative bursts in the first cycle. We have also modeled our previous results with this polymerase, including four situations with 8-oxo-7,8-dihydroguanine (8-oxoGua) moieties in which substoichiometric bursts (2-35%) were observed even after the correction of enzyme concentration by amino acid analysis. These include insertion of dATP opposite template 8-oxoGua, insertion of (deoxy) 8-oxoGua 5'-triphosphate opposite template C, and extension of primers beyond 8-oxoGua-A and 8-oxoGua-C pairs. The "minimal" polymerase mechanism and three others were evaluated using KINSIM and FITSIM methods. The latter three mechanisms involve a conformationally distinct, inactive polymerase-DNA-dNTP complex in equilibrium with the initial ternary complex and a conformationally distinct complex leading to phosphodiester bond formation. All three of the modified mechanisms fit the observed reaction results, but the minimal mechanism did not. Nonfunctional binary complexes (enzyme-DNA) are an alternate explanation (to ternary complexes) in some cases. Finally, DNA trapping experiments indicate that enzyme does not dissociate from the 8-oxoGua-containing DNA substrate prior to phosphodiester bond formation. We conclude that HIV-1 RT is fully active in normal nucleotide incorporation and that substoichiometric bursts with modified systems are well-described by the existence of nonproductive ternary complexes, which can isomerize to productive complexes.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Furge LL,Guengerich FPdoi
10.1021/bi982163usubject
Has Abstractpub_date
1999-04-13 00:00:00pages
4818-25issue
15eissn
0006-2960issn
1520-4995pii
bi982163ujournal_volume
38pub_type
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