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 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...

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

“…(27,42,44). The majority (13) of this group of 20 is involved in H-bonds to other solvent, and half make apolar OOH-C contacts.…”

“…It catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate, producing 5,6,7,8-THF. DHFR has been extensively studied by NMR (11), site-directed mutagenesis (12), and molecular dynamics (13) to unravel the potential coupling of motion by distal residues to catalysis (14,15). In particular, a regulatory loop, the Met-20 loop (residues 9-24), has been observed to adopt open, closed, and occluded conformations by x-ray crystallography (16) and closed and occluded conformations by NMR (17,18).…”

Allosteric Communication in Dihydrofolate Reductase: Signaling Network and Pathways for Closed to Occluded Transition and Back

“…Such a concept is appealing because the energy landscape of enzymes even in the folded state is rugged [5], and hence thermal energy might be sufficient to access several conformational substates during a typical reaction cycle [2]. In recent years, results from a number of studies have been used to propose that dynamic motions in a network of residues that promote catalytically-relevant structural transitions may be encoded in the protein structure [6,7,8,9,10,11,12,13,14,15,16]. While it is difficult to unambiguously demonstrate whether collective dynamics involving a network of residues facilitates catalysis [17], it is clear that enzymes sample a number of distinct states during a reaction cycle.…”

“…While it is difficult to unambiguously demonstrate whether collective dynamics involving a network of residues facilitates catalysis [17], it is clear that enzymes sample a number of distinct states during a reaction cycle. In the best studied example of E. coli dihydrofolate reductase (DHFR) the role of the conformational motions in the enzyme in facilitating the hydride transfer has been linked using mutational studies [10,18,19], NMR relaxation dispersion measurements [20,21,22] that probe the dynamics on µs to ms time scale, molecular dynamics simulations [6,7,13], and sequence analysis [6].…”

Computational delineation of the catalytic step of a high‐fidelity DNA polymerase

“…[24][25][26][27] There is evidence that such a coupling of fast and slow dynamical modes may play a significant role in DNA polymerase mechanisms. Pioneering single molecule studies of polymerases replicating DNA stretched under differing tensions 28,29 showed that the replication rate is sensitive to forces exerted on the template strand.…”