Network of coupled promoting motions in enzyme catalysis

Abstract

A network of coupled promoting motions in the enzyme dihydrofolate reductase is identified and characterized. The present identification is based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum/classical molecular dynamics simulations of hydride transfer. The motions in this network span time scales of femtoseconds to milliseconds and are found on the exterior of the enzyme as well as in the active site. This type of network has broad implications for an expanded role of the protein fold in catalysis as well as ancillaries such as the engineering of altered protein function and the action of drugs distal to the active site.

Figure 1

Secondary structure of DHFR. The Met-20 and βF–βG loops, as well as the NADPH coenzyme and DHF substrate, are labeled. All structural figures in this paper were generated by using the programs MOLSCRIPT and RASTER3D.

Figure 2

Sequence conservation in DHFR. Regions of conservation are mapped onto the structure of E. coli DHFR by using a gradient color scheme (gray to red, where red is the most conserved). NADPH and DHF are in green and magenta, respectively.

Figure 3

Diagram of a portion of the network of coupled promoting motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions.

Figure 4

Equilibrium averages of geometrical properties along the collective reaction coordinate. (A) Donor–acceptor distance. (B) Angle between the acceptor and methylene amino linkage in DHF. (C) DHF pterin ring puckering angle. (D) Distance between C_ζ of Phe-31 and C11 of DHF. (E) Hydrogen-bonding distance between N of Asp-122 and O of Gly-15 (red) and between O of Ile-14 and carboxamide N of NADPH (blue). (F) Distance between C_δ of Ile-14 and the side-chain oxygen of Tyr-100.

Figure 5

Time evolution of two select distances for a representative real-time vibrationally adiabatic trajectory. (A) Donor-acceptor distance. (B) Distance between C_α of Gly-121 and C_β of Met-42. Time t = 0 corresponds to the transition state, and the reaction evolves toward the reactant/product as the time becomes negative/positive. For B, the blue curve indicates a fit to a frequency on the picosecond time scale.