• 2018-07
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  • br Experimental section br Author contributions br Acknowled


    Experimental section
    Author contributions
    Introduction Many closantel
    in cells are sequential enzymatic reactions where the product of one enzyme serves as the substrate of a second enzyme (1, 2). Experimental evidence suggests that some of these consecutive enzyme-catalyzed reactions exploit substrate channeling to maximize the efficiency of the transport of product from the first active site to the second active site, at which the next enzyme-catalyzed reaction occurs (1). More precisely, substrate channeling refers to the scenario where an intermediate from one reaction site is transferred to a consecutive reaction site without complete mixing of the intermediate with the bulk solvent (3). This efficient transfer can be achieved through molecular tunnels, electrostatic channeling, or active sites in close proximity (3, 4). Although a molecular tunnel relies on the geometric confinement of intermediates to prevent their diffusion into bulk solvent, electrostatic-mediated substrate channeling utilizes electrostatic interactions to create a virtual tunnel that confines the intermediate between the two reaction sites (4, 5). A well-known example of electrostatic channeling along a solvent-exposed surface is the bifunctional protozoan dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme from Leishmania major. In this enzyme, the negatively charged (−2 net charge under physiological conditions) dihydrofolate intermediate synthesized at the TS active site subsequently reacts at the DHFR active site. Both kinetic experiments (6) and Brownian dynamics simulation studies (7) support the existence of electrostatic channeling of dihydrofolate between the two active sites of L. major DHFR-TS. The experimental evidence of substrate channeling is based on an observed decrease in the transient time for the final coupled enzyme product (in this case tetrahydrofolate) to appear relative to the time expected in a system without channeling, as well as an increased overall sensitivity of the net reaction rate to competitive inhibitors. Kinetic experiments on the bifunctional DHFR-TS enzyme from L. major DHFR-TS suggest that 80% or more of dihydrofolate molecules are channeled directly from the TS active site to the DHFR active site of this bifunctional enzyme (6). Brownian dynamics simulations performed in the past on L. major DHFR-TS also showed high transfer efficiency of intermediate that is >95% at zero ionic strength and >50% at physiological (150 mM) ionic strength (7). Although human DHFR and TS reactions are catalyzed by separate, monomeric enzymes, in some plants and protozoa, these two enzymes exist in a dimer structure with four active sites, including two TS active sites and two DHFR active sites (8, 9), as in L. major and Plasmodium falciparum (Fig. 1, A and B). Since the x-ray crystal structure of L. major DHFR-TS was determined, additional structures of bifunctional DHFR-TS enzymes from other protozoan species, such as P. falciparum DFHR-TS (8), Cryptosporidium hominis DHFR-TS (10), and Toxoplasma gondii DHFR-TS (11), have also been solved. Interestingly, these bifunctional protozoan enzymes share a common V-shaped geometry, with the main interface between the two monomers located at the bottom of the V shape where the TS domains intersect (Fig. 1). Because of the structural similarity between L. major DHFR-TS and other protozoan DHFR-TS enzymes, we hypothesized that these other enzymes may also support significant electrostatic channeling of dihydrofolate. However, despite multiple kinetic experiments investigating substrate channeling in this system, it has been found that the structurally similar DHFR-TS enzyme from C. hominis does not exhibit any measurable substrate channeling (12). This experimental result suggests that different protozoan DHFR-TS enzymes may exhibit varying efficiency of substrate channeling. In these systems, it appears that substrate channeling is dependent on the magnitude and position of important attractive electrostatic interactions between dihydrofolate and the enzyme, as well as on the geometry and proximity of the TS active site relative to the DHFR active site. For example, the distance between the TS and DHFR active sites of L. major DHFR-TS is shorter than the distance in P. falciparum and C. hominis DHFR-TS. Also, there is a greater density of basic, electropositive residues between the TS and DHFR active sites of L. major DHFR-TS compared to P. falciparum and C. hominis DHFR-TS.