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 quantum͞ 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.A relationship between the motion of protein structural elements and activity has been implicated in enzyme catalysis (1-3). Evidence for the existence of promoting vibrations or modes that augment catalytic activity has been sought for a number of enzymes. At the amino acid level, motions of residues both in and distal to the active site have been proposed to participate in catalysis. The identification, characterization, and clarification of the function of such proximal and distal promoting motions present a challenging task. Recently the importance of coupled motions sampled in differing time domains involving distal residues in the enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3) has been suggested by a combination of NMR experiments (microsecond to picosecond) (4), classical molecular dynamics simulations (nanosecond) (5), and kinetic experiments for site-directed mutants (millisecond to second) (6, 7). Here we report the results of genomic analysis, kinetic measurements of multiple mutations, and mixed quantum͞classical molecular dynamics simulations (8) of the hydride transfer step in DHFR. Based on the crystal structure framework, these results provide a description of specific residue motions and their linkage to enzyme catalysis.DHFR is required for normal folate metabolism in prokaryotes and eukaryotes. It catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) by using nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme. Specifically, the pro-R hydride of NADPH is transferred to the C6 of the pterin with concurrent protonation at the N5 position. This reaction is essential to maintain necessary levels of THF needed to support the biosynthesis of purines, pyrimidines, and amino acids, fostering DHFR as a pharmacological target. As a result of its importance, DHFR has been studied extensively with a wide range of methodologies.X-ray crystallographic studies indicate that the Escherichia coli DHFR enzyme contains an eight-stranded -sheet and four ␣-helices interspersed with flexible loop regions that connect these structural elements (ref. 9; see Fig. 1). Depending on the nature of the bound ligand, three different conformations have been observed for a surface loop formed by residues 9-24 (denoted the Met-20 loop). When the DHF substrate and NADPH coenzyme are bound, the Met-20...