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  • P falciparum DHFR TS is a particularly important enzyme

    2021-04-09

    P. falciparum DHFR-TS is a particularly important enzyme, because it is from the protozoan species that is associated with most malaria infections. Since this enzyme is responsible for catalyzing sequential reactions in the thymidylate cycle, its inhibition slows malarial dTMP production and therefore interferes with the process of DNA synthesis (8). Most antifolate antimalarial drugs act by inhibiting the DHFR activity of the P. falciparum enzyme (8), because there is greater structural variance between monomeric human DHFR and protozoan bifunctional DHFR domains compared to their very similar TS domains. However, because of growing drug resistance (8), strategies for designing novel PTC-209 of malarial drugs should be considered. If the transfer of the intermediate is highly dependent on electrostatic channeling, the disruption of this channeling could be an alternative strategy to selectively interfere with the thymidylate cycle in P. falciparum. P. falciparum DHFR-TS has been suspected as an enzyme that makes use of electrostatic channeling (8, 13). The wild-type crystal structure of P. falciparum DHFR-TS reveals a long, winding electropositive groove that connects the TS and DHFR active sites on each monomer of the bifunctional enzyme. As shown in Fig. 1 B, and first described by Yuvaniyama et al. (8), a groove exists in the solvent-accessible surface connecting the TS and DHFR active sites on each monomer, and the electrostatic representations show that the groove is positively charged. A second path, of lower electropositive magnitude and width, appears between the TS active site of one monomer and the DHFR active site of the other monomer (8). The electrostatic potential contour maps suggest that electrostatic channeling is possible because the TS and DHFR active sites are connected by regions of opposite charge to that of the channeled intermediate. As suggested by Eun et al. (4), the electrostatic attraction between active sites and intermediate is more influential in accelerating enzyme kinetics than the electrostatic guidance for an intermediate due to the existence of an electrostatic potential gradient between the two active sites. Also, mediators of charge opposite to that of the channeled intermediate can promote electrostatic channeling, even for well-separated active sites. Hence, we seek to determine whether the distribution of electropositive, solvent-exposed amino acids between the DHFR and TS active sites is sufficient for electrostatic-mediated dihydrofolate channeling. To quantitatively measure the strength of electrostatic channeling, we calculate the efficiency of transfer of newly synthesized dihydrofolate from the TS active site to the DHFR active site. A substrate channel that guides all of the intermediate synthesized at the first active site to the second active site would represent complete channeling efficiency (100% transfer efficiency), whereas diffusion of intermediate away from the protein to the bulk solution would result in transfer efficiencies approaching 0%. Because most instances of substrate channeling observed in nature are <100% (3), the transfer efficiency is an important quantity used to compare the relative leakiness of substrate channels. An objective of this study is to understand the magnitude of electrostatic channeling in wild-type P. falciparum DHFR-TS through theoretical methods using a variety of modeling approaches. To study the reaction kinetics, we employ Brownian dynamics simulations, and to calculate steady-state values, we rely on a PTC-209 continuum model approach. Although Brownian dynamics has been the computational method of choice for studying intermediate transfer efficiencies in systems suspected of electrostatic channeling, combining this approach with a continuum method allows us to explore the properties of the system more comprehensively and to generate the steady-state intermediate concentration maps on the enzyme’s solvent-accessible surface.