Dataset Information

19F-n.m.r. studies of 3',5'-difluoromethotrexate binding to Lactobacillus casei dihydrofolate reductase. Molecular motion and coenzyme-induced conformational changes.

ABSTRACT: 19F-n.m.r. spectroscopy was used to study the binding of 3',5'-difluoromethotrexate to dihydrofolate reductase (tetrahydrofolate dehydrogenase) from Lactobacillus casei. The benzoyl ring of the bound difluoromethotrexate was found to 'flip' about its symmetry axis, and the rate (7.3 X 10(3) s-1 at 298 K) and activation parameters for this process were determined by lineshape analysis of the 19F-n.m.r. spectrum at a series of temperatures in the range 273-308 K. The contributions to the barrier for this process are discussed. Addition of NADP+ or NADPH to form the enzyme-difluoromethotrexate-coenzyme ternary complex led to an increase in the rate of benzoyl ring flipping by a factor of 2.6-2.8-fold, and to substantial changes in the 19F-n.m.r. chemical shifts. The possible nature of the coenzyme-induced conformational changes responsible for these effects is discussed.

SUBMITTER: Clore GM

PROVIDER: S-EPMC1153266 | biostudies-other | 1984 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of (3)JCγCO and (3)JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as it proceeds through its enzymatic cycle, but that ligand flux is brokered by more subtle conformational changes that depend on the side chain motions of critical residues.

Dataset Information

19F-n.m.r. studies of 3',5'-difluoromethotrexate binding to Lactobacillus casei dihydrofolate reductase. Molecular motion and coenzyme-induced conformational changes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets