Explanation of Pre-Steady-State Kinetics and Decreased Burst Amplitude of HIV-1 Reverse Transcriptase at Sites of Modified DNA Bases with an Additional, Nonproductive Enzyme−DNA−Nucleotide Complex†

Biochemistry ◽  
1999 ◽  
Vol 38 (15) ◽  
pp. 4818-4825 ◽  
Author(s):  
Laura Lowe Furge ◽  
F. Peter Guengerich
Keyword(s):  
Steady State ◽  
Reverse Transcriptase ◽  
Dna Bases ◽  
Steady State Kinetics ◽  
Modified Dna ◽  
Burst Amplitude ◽  
Hiv 1

Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a β-thujaplicinol analogue

2013 ◽  
Vol 455 (2) ◽  
pp. 179-184 ◽  
Author(s):  
Brian D. Herman ◽  
Nicolas Sluis-Cremer
Keyword(s):  
Steady State ◽  
Reverse Transcriptase ◽  
Rnase H ◽  
Ribonuclease H ◽  
Cleavage Activity ◽  
Steady State Kinetics ◽  
Hiv 1

Pre-steady-state kinetics were used to define the mechanisms by which efavirenz and a β-thujaplicinol analogue modulate the RNase H activity of HIV-1 reverse transcriptase. Both inhibitors do not affect polymerase-dependent cleavages, but significantly affect the rates of polymerase-independent cleavages.

scholarly journals Steady state kinetics and inhibition of HIV-1 reverse transcriptase by a non-nucleoside dipyridodiazepinone, BI-RG-587, using a heteropolymeric template

1991 ◽  
Vol 19 (11) ◽  
pp. 3035-3039 ◽  
Author(s):  
Elizabeth B. Kopp ◽  
John J. Miglietta ◽  
Anthony G. Shrutkowski ◽  
Cheng-Kon Shih ◽  
Peter M. Grob ◽  
...  
Keyword(s):  
Steady State ◽  
Reverse Transcriptase ◽  
Steady State Kinetics ◽  
Hiv 1

scholarly journals Pre-steady-state Kinetic Studies Establish Entecavir 5′-Triphosphate as a Substrate for HIV-1 Reverse Transcriptase

2007 ◽  
Vol 283 (9) ◽  
pp. 5452-5459 ◽  
Author(s):  
Robert A. Domaoal ◽  
Moira McMahon ◽  
Chloe L. Thio ◽  
Christopher M. Bailey ◽  
Julian Tirado-Rives ◽  
...  
Keyword(s):  
Steady State ◽  
Reverse Transcriptase ◽  
Kinetic Studies ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Hiv 1

scholarly journals Mechanisms by Which the G333D Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Facilitates Dual Resistance to Zidovudine and Lamivudine

2007 ◽  
Vol 52 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Shannon Zelina ◽  
Chih-Wei Sheen ◽  
Jessica Radzio ◽  
John W. Mellors ◽  
Nicolas Sluis-Cremer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Steady State ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Steady State Kinetic ◽  
Immunodeficiency Virus ◽  
State Kinetic ◽  
Hiv 1

ABSTRACT Recent studies have identified a role for mutations in the connection and RNase H domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analog RT inhibitors (NRTI). To provide insight into the biochemical mechanism(s) involved, we investigated the effect of the G333D mutation in the connection domain of RT on resistance to zidovudine (AZT) and lamivudine (3TC) in enzymes that contain both M184V and thymidine analog mutations (TAMs; M41L, L210W, and T215Y). Our results from steady-state kinetic, pre-steady-state kinetic, and thermodynamic analyses indicate that G333D facilitates dual resistance to AZT and 3TC in two ways. First, in combination with M184V, G333D increased the ability of HIV-1 RT to effectively discriminate between the normal substrate dCTP and 3TC-triphosphate. Second, G333D enhanced the ability of RT containing TAMs and M184V to bind template/primer terminated by AZT-monophosphate (AZT-MP), thereby restoring ATP-mediated excision of AZT-MP under steady-state assay conditions. This study is the first to elucidate a molecular mechanism whereby a mutation in the connection domain of RT can affect NRTI susceptibility at the enzyme level.

scholarly journals Mechanism of Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase by a Stavudine Analogue, 4′-Ethynyl Stavudine Triphosphate

2008 ◽  
Vol 52 (6) ◽  
pp. 2035-2042 ◽  
Author(s):  
Guangwei Yang ◽  
Jimin Wang ◽  
Yao Cheng ◽  
Ginger E. Dutschman ◽  
Hiromichi Tanaka ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Steady State ◽  
Reverse Transcriptase ◽  
Structural Basis ◽  
Mechanism Of Inhibition ◽  
Steady State Kinetic ◽  
Immunodeficiency Virus ◽  
State Kinetic ◽  
Hiv 1

ABSTRACT 2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), a recently discovered nucleoside reverse transcriptase (RT) inhibitor, exhibits 5- to 10-fold-higher activity against human immunodeficiency virus type 1 (HIV-1) and less cytotoxicity than does its parental compound d4T (stavudine). Using steady-state kinetic approaches, we have previously shown that (i) 4′-ethynyl-d4T triphosphate (4′-Ed4TTP) inhibits HIV-1 RT more efficiently than d4TTP does and (ii) its inhibition efficiency toward the RT M184V mutant is threefold less than that toward wild-type (wt) RT. In this study we used pre-steady-state kinetic approaches in an attempt to understand its mechanism of inhibition. With wt and the M184V mutant RTs, 4′-Ed4TTP has three- to fivefold-lower Kd (dissociation constant) values than d4TTP, while d4TTP has up to eightfold-higher Kd values than dTTP. Inhibition is more effective in DNA replication with RNA template than with DNA template. In general, the M184V mutant exhibits poorer binding for all three nucleoside triphosphates than does wt RT. The structural basis for the lower binding affinity of d4TTP than of dTTP could be the lack of hydrogen bonds from the missing 3′-hydroxyl group in d4TTP to the backbone amide of Y115 and also to the side chain of Q151. The structural basis for the higher binding affinity of 4′-Ed4TTP than of d4TTP could be the additional binding of the 4′-ethynyl group in a preformed hydrophobic pocket by A114, Y115, M184, F160, and part of D185.

scholarly journals Nonnucleoside Reverse Transcriptase Inhibitor Hypersusceptibility and Resistance by Mutation of Residue 181 in HIV-1 Reverse Transcriptase

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
John P. Barnard ◽  
Kelly D. Huber ◽  
Nicolas Sluis-Cremer
Keyword(s):  
Reverse Transcriptase ◽  
Transcriptase Inhibitor ◽  
Cross Resistance ◽  
Wild Type ◽  
Nonnucleoside Reverse Transcriptase Inhibitor ◽  
Steady State Kinetics ◽  
Deoxynucleoside Triphosphate ◽  
Reverse Transcriptase Inhibitor ◽  
Insight Into ◽  
Hiv 1

ABSTRACT Substitutions at residue Y181 in HIV-1 reverse transcriptase (RT), in particular, Y181C, Y181I, and Y181V, are associated with nonnucleoside RT inhibitor (NNRTI) cross-resistance. In this study, we used kinetic and thermodynamic approaches, in addition to molecular modeling, to gain insight into the mechanisms by which these substitutions confer resistance to nevirapine (NVP), efavirenz (EFV), and rilpivirine (RPV). Using pre-steady-state kinetics, we found that the dissociation constant (Kd) values for inhibitor binding to the Y181C and Y181I RT-template/primer (T/P) complexes were significantly reduced. In the presence of saturating concentrations of inhibitor, the Y181C RT-T/P complex incorporated the next correct deoxynucleoside triphosphate (dNTP) more efficiently than the wild-type (WT) complex, and this phenotype correlated with decreased mobility of the RT on the T/P substrate. Interestingly, we found that the Y181F substitution in RT—which represents a transitional mutation between Y181 and Y181I/V, or a partial revertant—conferred hypersusceptibility to EFV and RPV at both the virus and enzyme levels. EFV and RPV bound more tightly to Y181F RT-T/P. Furthermore, inhibitor-bound Y181F RT-T/P was less efficient than the WT complex in incorporating the next correct dNTP, and this could be attributed to increased mobility of Y181F RT on the T/P substrate. Collectively, our data highlight the key role that Y181 in RT plays in NNRTI binding.

scholarly journals Pre-steady state kinetic analysis of cyclobutyl derivatives of 2′-deoxyadenosine 5′-triphosphate as inhibitors of HIV-1 reverse transcriptase

2012 ◽  
Vol 22 (12) ◽  
pp. 4064-4067 ◽  
Author(s):  
Jiae Kim ◽  
Ligong Wang ◽  
Yongfeng Li ◽  
Kimberlynne D. Becnel ◽  
Kathleen M. Frey ◽  
...  
Keyword(s):  
Steady State ◽  
Reverse Transcriptase ◽  
Kinetic Analysis ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Derivatives Of ◽  
Hiv 1
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