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Mass-dependent bond vibrational dynamics influence catalysis by HIV-1 protease.

ABSTRACT: Protein motions that occur on the microsecond to millisecond time scale have been linked to enzymatic rates observed for catalytic turnovers, but not to transition-state barrier crossing. It has been hypothesized that enzyme motions on the femtosecond time scale of bond vibrations play a role in transition state formation. Here, we perturb femtosecond motion by substituting all nonexchangeable carbon, nitrogen, and hydrogen atoms with (13)C, (15)N, and (2)H and observe the catalytic effects in HIV-1 protease. According to the Born-Oppenheimer approximation, isotopic substitution alters vibrational frequency with unchanged electrostatic properties. With the use of a fluorescent peptide to report on multiple steps in the reaction, we observe significantly reduced rates in the heavy enzyme relative to the light enzyme. A possible interpretation of our results is that there exists a dynamic link between mass-dependent bond vibrations of the enzyme and events in the reaction coordinate.

SUBMITTER: Kipp DR

PROVIDER: S-EPMC3227791 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Mass-dependent bond vibrational dynamics influence catalysis by HIV-1 protease.

Kipp D Randal DR Silva Rafael G RG Schramm Vern L VL

Journal of the American Chemical Society 20111111 48

Protein motions that occur on the microsecond to millisecond time scale have been linked to enzymatic rates observed for catalytic turnovers, but not to transition-state barrier crossing. It has been hypothesized that enzyme motions on the femtosecond time scale of bond vibrations play a role in transition state formation. Here, we perturb femtosecond motion by substituting all nonexchangeable carbon, nitrogen, and hydrogen atoms with (13)C, (15)N, and (2)H and observe the catalytic effects in H ...[more]

PMID: 22059645

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Project description:BACKGROUND:HIV-1 can develop resistance to antiretroviral drugs, mainly through mutations within the target regions of the drugs. In HIV-1 protease, a majority of resistance-associated mutations that develop in response to therapy with protease inhibitors are found in the protease's active site that serves also as a binding pocket for the protease inhibitors, thus directly impacting the protease-inhibitor interactions. Some resistance-associated mutations, however, are found in more distant regions, and the exact mechanisms how these mutations affect protease-inhibitor interactions are unclear. Furthermore, some of these mutations, e.g. N88S and L76V, do not only induce resistance to the currently administered drugs, but contrarily induce sensitivity towards other drugs. In this study, mutations N88S and L76V, along with three other resistance-associated mutations, M46I, I50L, and I84V, are analysed by means of molecular dynamics simulations to investigate their role in complexes of the protease with different inhibitors and in different background sequence contexts. RESULTS:Using these simulations for alchemical calculations to estimate the effects of mutations M46I, I50L, I84V, N88S, and L76V on binding free energies shows they are in general in line with the mutations' effect on [Formula: see text] values. For the primary mutation L76V, however, the presence of a background mutation M46I in our analysis influences whether the unfavourable effect of L76V on inhibitor binding is sufficient to outweigh the accompanying reduction in catalytic activity of the protease. Finally, we show that L76V and N88S changes the hydrogen bond stability of these residues with residues D30/K45 and D30/T31/T74, respectively. CONCLUSIONS:We demonstrate that estimating the effect of both binding pocket and distant mutations on inhibitor binding free energy using alchemical calculations can reproduce their effect on the experimentally measured [Formula: see text] values. We show that distant site mutations L76V and N88S affect the hydrogen bond network in the protease's active site, which offers an explanation for the indirect effect of these mutations on inhibitor binding. This work thus provides valuable insights on interplay between primary and background mutations and mechanisms how they affect inhibitor binding.

Dataset Information

Mass-dependent bond vibrational dynamics influence catalysis by HIV-1 protease.

Publications

Mass-dependent bond vibrational dynamics influence catalysis by HIV-1 protease.

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