Evolutionary Effects on Bound Substrate pK_a in Dihydrofolate Reductase

Abstract

In the present study, we address the effect of active site structure and dynamics of different dihydrofolate reductase (DHFR) isoforms on the pK a of the bound substrate 7,8-dihydrofolate, in an attempt to understand possible evolutionary trends. We apply a hybrid QM/MM free energy perturbation method to estimate the pK a of the N5 position of the bound substrate. We observe a gradual increase in N5 basicity as we move from primitive to more evolved DHFR isoforms. Structural analysis of these isoforms reveals a gradual sequestering of water molecules from the active site in the more evolved enzymes, thereby modulating the local dielectric environment near the substrate. Furthermore, the present study reveals a clear correlation between active site hydration and the N5 pK a of the substrate. We emphasize the role of the M20 loop in controlling the active site hydration level, via a preorganized active site with a more hydrophobic environment and reduced loop flexibility as evolution progresses from bacterial to the human enzyme.

Thermal Adaptation of Enzymes: Impacts of Conformational Shifts on Catalytic Activation Energy and Optimum Temperature

“…The Met20 loop has been suggested to play two important roles during the catalyzed hydride transfer: i) keeping the folate substrate next to the cofactor and ii) protecting the substrate reactive N5 site (Figure c) from the polar aqueous solvent . These two aspects can be best understood using a theoretical model that established that hydride‐ (and proton‐) transfer free energy barriers sensitively depend on the rearrangement of two coordinates: first, the electrostatic environment which reorganizes to stabilize the nascent product charge distribution, and second the contraction of the hydride donor–acceptor distance (here CC) which facilitates the transfer (the third coordinate is the displacement of the hydride particle, which adapts very fast to the two other slower coordinates).…”

DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents

“…To date, the Protein Data Bank (PDB) has collected over one hundred structures obtained from both eukaryotic and prokaryotic organisms (humans, Escherichia coli , Lactobacillus casei , Pneumocystis carinii , Micobacterium tuberculosis , etc. ), alone or in complex with different ligands [16]. Briefly, DHFR consists of eight sheets, which form a rigid skeleton: seven sheets run parallel and the other runs antiparallel.…”

Multiscale simulations identify origins of differential carbapenem hydrolysis by the OXA-48 β-lactamase

“…To avoid or limit the occurrence of additional hydrogen bonding to catalytic bases, enzymes have evolved active site architectures that can promote desolvation to increase carboxylate reactivity. Such desolvation can for example be achieved by loop closure (as in triosephosphate isomerase and dihydrofolate reductase) 63,64 or closure of the substrate binding cleft (as in ketosteroid synthase). Here, subtle control of the solvation around the carboxylated Lys73 is related to nearby hydrophobic residues (Val120 and Leu158), which can adopt conformations that allow the presence of the deacylating water but avoid more extensive solvation of the catalytic carboxylate.…”