Methotrexate complex with DHFR

Figure 6. Methotrexate complexed with dihydrofolate reductase (DHFR). Dotted surface represents extended VDW surface for the DHFR active site residues. Methotrexate heteroatoms are labelled...

The power of NMR methods for studying dynamics of complexes is beautifully illustrated by an earlier study of the complex of DHFR with methotrexate.147 In this case a correlated dynamic rotation of a carboxylate group on the ligand and Arg57 of the protein was detected as illustrated in Fig. 28. 

The method was tested by attempting to reproduce the bound conformations of two enzyme-inhibitor complexes Streptomyces griseus proteinase B (SGPB) complexed with the ovomucoid inhibitor from turkey (OMTKY3), and dihydrofolate reductase (DHFR) complexed with methotrexate (MTX). The... 

We have already used the interactions of methotrexate with dihydrofolate reductase (DHFR) several times within this text to illustrate some key aspects of enzyme inhibition. The reader will recall that methotrexate binds to both the free enzyme and the enzyme-NADPH binary complex but displays much greater affinity for the latter species. The time dependence of methotrexate binding to bacterial DHFR was studied by Williams et al. (1979) under conditions of saturating [NADPH], In the presence of varying concentrations of methotrexate, the progress curves for DHFR activity became progressively more nonlinear (Figure 6.14). The value of kobs from... 

E. coli enzyme. But, studies on the chicken liver enzyme may not be relevant to the human enzyme. Indeed, crystal studies of Oefner et al. (1988) on complexes of human DHFR with folate, methotrexate and trimethoprim itself do not support the explanation given by Matthews, and so we are still not sure of the basis for the species selectivity exhibited by trimethoprim. 

Figure 12.28. Correlated motions of a carboxylate group from methotrexate and Arg57 of DHFR detected by NMR (a) Structure of an arginine-car-boxylate complex formed with symmetrical end-on interactions and (b) structure of methotrexate showing its interaction with the guanidine group of Arg57 of DHFR. (Reprinted with permission from Ref 133.)...

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an essential enzyme required for normal folate metabolism in prokaryotes and eukaryotes. Its role is to maintain necessary levels of tetrahydrofolate to support the biosynthesis of purines, pyrimidines and amino acids. Many compounds of pharmacological value, notably methotrexate and trimethoprim, vork by inhibition of DHFR. Their clinical importance justified the study of DHFR in the rapidly evolving field of enzymology. Today, there is a vast amount of published literature (ca. 1000 original research articles) on the broad subject of dihydrofolate reductase contributed by scientists from diverse disciplines. We have selected kinetic, structural, and computational studies that have advanced our understanding of the DHFR catalytic mechanism with special emphasis on the role of the enzyme-substrate complexes and protein motion in the catalytic efficiency achieved by this enzyme. 

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