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Pre-steady-state kinetic studies establish entecavir 5'-triphosphate as a substrate for HIV-1 reverse transcriptase.

ABSTRACT: The novel 2'-deoxyguanosine analog Entecavir (ETV) is a potent inhibitor of hepatitis B virus (HBV) replication and is recommended for treatment in human immunodeficiency virus type 1 (HIV-1) and HBV-co-infected patients because it had been reported that ETV is HBV-specific. Recent clinical observations, however, have suggested that ETV may indeed demonstrate anti-HIV-1 activity. To investigate this question at a molecular level, kinetic studies were used to examine the interaction of 5'-triphosphate form of ETV with wild type (WT) HIV-1 reverse transcriptase (RT) and the nucleoside reverse transcriptase inhibitor-resistant mutation M184V. Using single turnover kinetic assays, we found that HIV-1 WT RT and M184V RT could use the activated ETV triphosphate metabolite as a substrate for incorporation. The mutant displayed a slower incorporation rate, a lower binding affinity, and a lower incorporation efficiency with the 5'-triphosphate form of ETV compared with WT RT, suggesting a kinetic basis for resistance. Our results are supported by cell-based assays in primary human lymphocytes that show inhibition of WT HIV-1 replication by ETV and decreased susceptibility of the HIV-1 containing the M184V mutation. This study has important therapeutic implications as it establishes ETV as an inhibitor for HIV-1 RT and illustrates the mechanism of resistance by the M184V mutant.

SUBMITTER: Domaoal RA

PROVIDER: S-EPMC7748947 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Publications

Breathlessness and palliative oxygen therapy in advanced chronic obstructive pulmonary disease.

Smallwood Natasha N Gaffney Nicole N Gorelik Alexandra A Irving Louis L Le Brian B Philip Jennifer J

Internal medicine journal 20180401 4

PMID: 29623984

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Project description:Isomer-specific 3-chloroacrylic acid dehalogenases catalyze the hydrolytic dehalogenation of the cis- and trans-isomers of 3-chloroacrylate to yield malonate semialdehyde. These reactions represent key steps in the degradation of the nematocide, 1,3-dichloropropene. The kinetic mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) has now been examined using stopped-flow and chemical-quench techniques. Stopped-flow analysis of the reaction, following the fluorescence of an active site tryptophan, is consistent with a minimal three-step model involving substrate binding, chemistry, and product release. Chemical-quench experiments show burst kinetics, indicating that product release is at least partially rate limiting. Global fitting of all of the kinetic results by simulation is best accommodated by a four-step mechanism. In the final kinetic model, the enzyme binds substrate with an immediate isomerization to an alternate fluorescent form and chemistry occurs, followed by the ordered release of two products, with the release of the first product as the rate-limiting step. Bromide ion is a competitive inhibitor of the reaction indicating that it binds to the free enzyme rather than to the enzyme with one product still bound. This observation suggests that malonate semialdehyde is the first product released by the enzyme (rate limiting), followed by halide. A comparison of the unliganded cis-CaaD crystal structure with that of an inactivated cis-CaaD where the prolyl nitrogen of Pro-1 is covalently attached to (R)-2-hydroxypropanoate provides a possible explanation for the isomerization step. The structure of the covalently modified enzyme shows that a seven-residue loop comprised of residues 32-38 is closed down on the active site cavity where the backbone amides of two residues (Phe-37 and Leu-38) interact with the carboxylate group of the adduct. In the unliganded form, the same loop points away from the active site cavity. Similarly, substrate binding may cause this loop to close down on the active site and sequester the reaction from the external environment.

Project description:DNA and RNA polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. We previously showed, for the reverse transcriptase (RT) encoded by the yeast retrotransposon Ty1, that one of the three conserved active site aspartates (D(211)) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. Transposition is partially restored by second site suppressor mutations in the RNAse H domain. The novel properties of this amino acid substitution led us to express the WT and D(211)N mutant enzymes, and study their pre-steady state kinetic parameters. We found that the k(pol) was reduced by a factor of 223 in the mutant, although the K(d) for nucleotide binding was unaltered. Further, the mutant enzyme had a marked preference for Mn(2+) over Mg(2+). To better understand the functions of this residue within the Ty1 RT active site, we have now examined the in vitro properties of WT and D(211)N mutant Ty1 RTs in carrying out pyrophosphorolysis, the reverse reaction to polymerization, where pyrophosphate is the substrate and dNTPs are the product. We find that pyrophosphorolysis is efficient only when the base-paired primer template region is >14 bases, and that activity increases when the primer end is blunt-ended or recessed by only a few bases. Using pre-steady state kinetic analysis, we find that the rate of pyrophosphorolysis (k(pyro)) in the D(211)N mutant is nearly 320 fold lower than the WT enzyme, and that the mutant enzyme has an approximately 170 fold lower apparent K(d) for pyrophosphate. These findings indicate that subtle substrate differences can strongly affect the enzyme's ability to properly position the primer-end to carry out pyrophosphorolysis. Further the kinetic data suggests that the D(211) residue has a role in pyrophosphate binding and release, which could affect polymerase translocation, and help explain the D(211)N mutant's transposition defect.

Dataset Information

Pre-steady-state kinetic studies establish entecavir 5'-triphosphate as a substrate for HIV-1 reverse transcriptase.

Publications

Breathlessness and palliative oxygen therapy in advanced chronic obstructive pulmonary disease.

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