Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease

Agnieszka Krzemińska, Vicent Moliner, Katarzyna Świderek
2016 Journal of the American Chemical Society  
HIV-1 Protease (HIV-1 PR) is one of the three enzymes essential for the replication process of HIV-1 virus, which explains why it has been the main target for design of drugs against acquired immunodeficiency syndrome (AIDS). This work is focused on exploring the proteolysis reaction catalyzed by HIV-1 PR, with special attention to the dynamic and electrostatic effects governing its catalytic power. Free energy surfaces for all possible mechanisms have been computed in terms of potentials of
more » ... n force (PMFs) within hybrid QM/MM potentials, with the QM sub-set of atoms described at semiempirical (AM1) and DFT (M06-2X) level. The results suggest that the most favorable reaction mechanism involves formation of a gem-diol intermediate, whose decomposition into the product complex would correspond to the rate-limiting step. The agreement between the activation free energy of this step with experimental data, as well as kinetic isotope effects (KIEs), supports this prediction. The role of the protein dynamic was studied by protein isotope labelling in the framework of the Variational Transition State Theory. The predicted enzyme KIEs, also very close to the values measured experimentally, reveal a measurable but small dynamic effect. Our calculations show how the contribution of dynamic effects to the effective activation free energy appears to be below 1 kcal·mol −1 . On the contrary, the electric field created by the protein in the active site of the enzyme emerges as being critical for the electronic reorganization required during the reaction. These electrostatic properties of the active site could be used as a mould for future drug design. GRAPHICAL ABSTRACT SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: *** Empirical estimation of pKa values calculated with PropKa; averaged values of KIE, BIE and EIEs obtained at AM1/MM level; Cartesian coordinates of QM subset of atoms for all TS structures optimized at M06-2X/MM level; AM1/MM PMFs obtained for all explored chemical steps; key interatomic distances and atomic charges of stationary point structures located and refined at M06-2X/MM level of theory; electric field created by the protein and water molecules in the center of the active site and in the C and N atoms of the scissile peptide bond; normalized autocorrelation function for light and heavy enzyme for two localized transition states TS3 and TS4 obtained at AM1/MM level; PMFs obtained in the vicinity of the transition state for two localized transition states TS3 and TS4 obtained at AM1/MM level. Keywords HIV-1 protease; proteolysis; peptide bond; KIEs; EIE; enzyme KIE; QM/MM; dynamic effects; heavy enzymes long-time the most accepted mechanism. It assumes that the water molecule is held and Krzemińska et al. Part of the uncertainty of the mechanism of HIV-1 PR is linked to the debate of the protonation state of Asp25(A) and Asp25(B). In fact, all four possibilities have been taken into consideration in the past. Meek and co-workers 17 demonstrated that pK a values are different for Asp25(A) and Asp25(B), and equal to 3.1 and 5.1, respectively, concluding that only one of them should be protonated. On the other hand, Kent and co-workers 37 claimed that both residues are negatively charged at pH = 6 in the absence of an inhibitor, while Parrinello and co-workers, 38 based on computational results, suggested that both residues should be protonated. Nevertheless, from the first experimental evidence of Torchia and coworkers, 39 it is now well accepted, in agreement with a broad range of aspartic proteases, that the aspartic groups are in opposite states of protonation, such that the catalytically competent form of these enzymes is a "mono-protonated" one. This conclusion is based on Krzemińska et al.
doi:10.1021/jacs.6b06856 pmid:27935692 pmcid:PMC5178878 fatcat:xdlhjr2645cwndwktoiujhgi3u