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Role of induced fit in limiting discrimination against AZT by HIV reverse transcriptase.

ABSTRACT: Single turnover kinetic studies were conducted using fluorescently labeled HIV reverse transcriptase (RT) to evaluate the role of nucleotide-induced changes in enzyme structure in the selectivity against AZT in order to explore why AZT-resistant forms of the enzyme fail to significantly discriminate against AZT. Fluorescent labeling of HIV RT provided a signal to monitor the isomerization from "open" to "closed" states following nucleotide binding. We measured the rate constants governing nucleotide binding and enzyme isomerization for TTP and AZT-triphosphate by the wild-type and AZT-resistant forms of the enzyme containing the thymidine analogue mutations (TAMs). We show that the TAMs alter the kinetics of AZT incorporation by weakening ground-state nucleotide binding and decreasing the rate of chemistry relative to the wild-type enzyme. However, the slower rate of incorporation of AZT by the TAMs HIV RT is counterbalanced a lower K(m), resulting from the equilibration of the conformational change step. In contrast, the K(m) for the wild-type enzyme reflects the balance between rates of binding and incorporation so the conformational change step does not come to equilibrium. These data once again demonstrate that the rate of substrate release, limited by the reverse of the substrate-induced conformational change, is the key determinant of the role of induced fit in enzyme specificity. Mutations leading to slower rates of incorporation have the unfortunate consequence of lowering the K(m) value by allowing the conformational change step to come to equilibrium.

SUBMITTER: Kellinger MW

PROVIDER: S-EPMC3381507 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Role of induced fit in limiting discrimination against AZT by HIV reverse transcriptase.

Kellinger Matthew W MW Johnson Kenneth A KA

Biochemistry 20110513 22

Single turnover kinetic studies were conducted using fluorescently labeled HIV reverse transcriptase (RT) to evaluate the role of nucleotide-induced changes in enzyme structure in the selectivity against AZT in order to explore why AZT-resistant forms of the enzyme fail to significantly discriminate against AZT. Fluorescent labeling of HIV RT provided a signal to monitor the isomerization from "open" to "closed" states following nucleotide binding. We measured the rate constants governing nucleo ...[more]

PMID: 21548586

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Project description:The replication of simian foamy virus from macaques can be inhibited by the nucleoside reverse transcriptase inhibitor azidothymidine (AZT, zidovudine). Four substitutions in the protease-reverse transcriptase (PR-RT) protein (K211I, I224T, S345T, E350K) are necessary to obtain highly AZT resistant and fully replication competent virus. AZT resistance is based on the excision of the incorporated AZTMP in the presence of ATP. I224T is a polymorphism which is not essential for AZT resistance per se, but is important for regaining efficient replication of the resistant virus.We constructed PR-RT enzymes harboring one to four amino acid substitutions to analyze them biochemically and to determine their ability to remove the incorporated AZTMP. S345T is the only single substitution variant exhibiting significant AZTMP excision activity. Although K211I alone showed no AZTMP excision activity, excision efficiency doubled when K211I was present in combination with S345T and E350K. K211I also decreased nucleotide binding affinity and increased fidelity. NMR titration experiments revealed that a truncated version of the highly AZT resistant mt4 variant, comprising only the fingers-palm subdomains was able to bind ATP with a KD-value of ca. 7.6 mM, whereas no ATP binding could be detected in the corresponding wild type protein. We could show by NMR spectroscopy that S345T is responsible for ATP binding, probably by making a tryptophan residue accessible.Although AZT resistance in SFVmac is based on excision of the incorporated AZTMP like in HIV-1, the functions of the resistance substitutions in SFVmac PR-RT appear to be different. No mutation resulting in an aromatic residue like F/Y215 in HIV, which is responsible for ?-?-stacking interactions with ATP, is present in SFVmac. Instead, S345T is responsible for creating an ATP binding site, probably by making an already existing tryptophan more accessible, which in turn can interact with ATP. This is in contrast to HIV-1 RT, in which an ATP binding site is present in the WT RT but differs from that of the AZT resistant enzyme.

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Role of induced fit in limiting discrimination against AZT by HIV reverse transcriptase.

Publications

Role of induced fit in limiting discrimination against AZT by HIV reverse transcriptase.

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