Mg²⁺ Binds to the Surface of Thymidylate Synthase and Affects Hydride Transfer at the Interior Active Site

Abstract

Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e. the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg2+ has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg2+ binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg2+ near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg2+ binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg2+ to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg2+ accelerates the hydride transfer by ca. 7-fold but does not affect the magnitude or temperature-dependence of the intrinsic kinetic isotope effect. These results suggest that Mg2+ facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg2+ concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg2+ with the glutamyl-tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with poly-glutamyl substitutes as species-specific antibiotic drugs.

Kinetic isotope effects as a probe of hydrogen transfers to and from common enzymatic cofactors

“…We have recently studied the ion’s mechanism of action through comprehensive kinetic, structural, and dynamics analyses [72]. Fluorescence assays, crystal structures, and NMR chemical shift changes show that Mg 2+ loosely binds (K d =3.7 mM) to the surface of TSase and forms a bridge between the glutamyl moiety of the folate cofactor and the enzyme through an H-bonding network.…”

“…(C) Change in chemical shift of each residue shown from white (no change) to red (maximum change). Reproduced from ref [72] with permission from ACS.…”

Catalytic mechanisms of thymidylate synthases

“…Furthermore, both our experiments and computations suggest TSase exploits concerted protein motions in catalyzing chemical transformations. [65][66][67][68] Particularly, our experiments with Y209W ecTSase demonstrated that protein motions at various time calculations. 23,108,[130][131][132] Compared with the extensively studied "model protein" DHFR, most enzymes are more similar to TSase, which have more rigid structures and sophisticated mechanisms.…”

“…65 Chapter IV extends this methodology to investigate the effects of Mg 2+ on the catalytic mechanism of TSase, which complements the studies in Chapter III and highlights the importance of long-range interactions in TSase-catalyzed reaction. 66 Chapter V presents our QM/MM calculations on the proton transfer step and compares the results with the previously calculated hydride transfer step, providing molecular details underlying the experimental KIE results. 67 It is noteworthy that our computational results not only illustrate functionalities of specific protein residues that reconcile many previous experimental observations, but also provide new insights into the catalytic mechanism that can be examined by future experiments.…”

“…We found that the stability of CH 2 H 4 folate is dependent on its own concentration, and are currently performing molecular dynamics (MD) simulations to understand this phenomenon. 220 The experimental-theoretical interactive approach taken in this project has not only assisted in understanding the catalytic mechanisms of TSase, but also provided insights into the relationship between protein structures, motions, and catalytic activity. We also studied the effects of Mg 2+ on the catalytic mechanism of TSase, which is the first attempt to resolve the mechanism of interaction between Mg 2+ and TSase.…”