Abstract
The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.
Highlights
Where E is Reverse transcriptase (RT), Dn,n+isl primer/template, dNTPis deoxyribonucleoside triphosphate, andPPi is pyrophosphate
Reverse transcriptase (RT)’ of human immunodeficiency virus 1 (HIV-1) possesses three catalytic activities that are essential for viralreplication [1, 2]: RNA-dependentDNA polymerase, RNase H, and DNA-dependent DNpAolymerase [3].The virion enzyme is a heterodimer of a 66-kDa polypeptide(p66)and a 51-kDa polypeptide (p51)thatis derived from p66 by proteolysis within the COOH-terminal portion [4,5,6]
This paper describes a morecomplete analysis of the DNA-dependent polymerase activity based on the analysoisf both theforward and reverse reactions
Summary
Lifetimes inthe absence conductivity equal to thoaft buffer A containing 50 mM KC1 (Fraction of M$+ were measured in the chemical quench apparatus asfollows: 111; 640 ml, 2.2 g of protein; 1.1 pmol min" mg"). R T activity lution (220 pl) of d T T P (300 p M ) and MgC1, (6 mM) inreaction eluted a t 0.2 M KC1 (Fraction IV; 340 ml, 469 mg of protein; 2.0 pmol buffer.After further incubation (10-40 s) at 22 "C, reactions were min" mg"). Centrifugation (16,000 X g X 30 rnin), the precipitate (150 mg) was 32-mer (200nM) for 10 min inthe presence of 6 mM MgC12in reaction dissolved in 10 ml of buffer C Collection by centrifugation (16,000 X g X 30 min), the precipitate mer (250 nM) and MgC1, (6 mM) in the reaction buffer were mixed was frozen in aliquots for long term storage at -70 "C.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
