PMC

(A) Schematic of reactions catalyzed by bifunctional TS-DHFR and substrate channeling. The TS reaction converts CH₂H₄F and dUMP to H₂F and dTMP, while the DHFR reaction converts H₂F to H₄F using NADPH as a cofactor. Substrate channeling occurs when H₂F is directly transferred from the TS to the DHFR without being released into solution by the TS and then bound from solution by DHFR. (B) Interspecies comparison of domains and linker regions in TS-DHFR.

Diagram of the pulse-chase experiment for measuring H₂F channeling. We expected a channeling enzyme (A) to directly transfer the radiolabeled H₂F produced by the TS reaction to the DHFR domain without releasing it into solution, therefore only a small amount of the H₂F intermediate product would be observed. However, an enzyme unable to channel (B) would release radiolabeled H₂F into solution, where it would be outcompeted by an excess of unlabeled H₂F for substrate binding sites in the DHFR domain. In this scenario, a substantial buildup of radiolabeled H₂F in solution would be observed.

Representative stopped-flow trace of DHFR catalysis of (A) WT and (B) W296A mutant as measured by NADPH fluorescence energy transfer at 450 nM. Enzyme (50 μM) was incubated with 250 μM of NADPH and mixed with 25 μM H₂F to examine the rate of chemistry (k_chem) in a single enzyme turnover. The data were to a single exponential for determine the rate of NADP⁺ product formation.

Coulombic surface charge distributions of channeling and non-channeling TS-DHFRs. Figures of coloumbic charges were created by using Chimera⁽⁾ using default settings. Negative charges are colored red, and positive charges are colored blue. Pictured are the TS-DHFRs from (A) T. gondii (4EIL, loop truncated mutant), (B) L. major⁽⁾, (C) P. falciparum (PDB ID: 1J3I), and (D) C. hominis (PDB ID: 1QZF). The DHFR domain of each enzyme is circled. An asterisk (*) indicates a version of TS-DHFR capable of channeling.

(A) Overall crystal structure of the T. gondii TS-DHFR. The TS domains for each monomer are highlighted in red and pink, the DHFR domains are in blue and cyan, and the junctional regions are in two shades of green. The flexible surface loop 1 and 3 deletions are denoted by dashed lines (shown only for the DHFR domain in blue) (B) The kinked crossover helix and its position between the DHFR and the TS domains. (C) Key hydrophobic interactions between the crossover helix and the adjacent DHFR domain.

Single turnover pulse-chase bifunctional reaction time courses for (A) T. gondii and (B) C. hominis TS-DHFR fit to a single exponential curve, showing the consumption of CH₂H₄F (black triangles) and production of H₄F (blue circles). Concentrations were normalized assuming the three substrates had a sum of 100% total content. Shown are representative time courses of experiments that were repeated in triplicate to confirm the reproducibility of the resultant rates. The peak of dihydrofolate buildup (red squares) is indicated by arrows.