Dihydrofolate reductase , binding

A complete kinetic scheme has been established for the enzyme from both sources. The L. casei dihydrofolate reductase followed a reaction sequence identical to the E. coli enzyme (Scheme I) moreover, none of the rate constants varied by more than 40-fold Figure 20 is a reaction coordinate diagram comparing the steady-state turnover pathway for E. coli and L. casei dihydrofolate reductase, drawn at an arbitrary saturating concentration (1 mM) of NADPH at pH 7. The two main differences are (i) L. casei dihydrofolate reductase binds NADPH more tightly in both binary (E-NH, -2 kcal/mol) and tertiary (E NH-H2F, - 1.4 kcal/mol E-NH-H4F, - 1.8 kcal/mol) complexes, and (ii) the internal equilibrium constant (E-NH H2F E-N-H4F) for hydride transfer is less favorable for the L. casei enzyme (1 kcal/mol). These changes, as noted later, are smaller than those observed for single amino acid substitutions at the active site of either enzyme. Thus, the overall kinetic sequence as well as the... 

The second type of antifolates bind preferentially with, and thus selectively inhibit, the enzyme dihydrofolate reductase contained in the plasmodia. This interferes with the abiUty of the malaria parasites to convert dihydrofolate to tetrahydrofoUc acid. In the erythrocyte host, however, dihydrofolate... 

A review is given of the application of Molecular Dynamics (MD) computer simulation to complex molecular systems. Three topics are treated in particular the computation of free energy from simulations, applied to the prediction of the binding constant of an inhibitor to the enzyme dihydrofolate reductase the use of MD simulations in structural refinements based on two-dimensional high-resolution nuclear magnetic resonance data, applied to the lac repressor headpiece the simulation of a hydrated lipid bilayer in atomic detail. The latter shows a rather diffuse structure of the hydrophilic head group layer with considerable local compensation of charge density. 

Matthews DA, Alden RA, Freer ST, Nguyen HX, Kraut J. Dihydrofolate reductase txom Lactobacillus casei. Stereochemistry of NADPH binding./Rio/ Chem 1979 254 4144-51. 

Kuyper LF, Roth B, Baccanari DP, Ferone R, Beddell CR, Champness JN et al. Receptor-based design of dihydrofolate reductase inhibitors comparison of crys-tallographically determined enzyme binding with enzyme affinity in a series of carboxy-substituted trimethoprim analogues. J Med Chem 1982 25 1120-2... 

Figure 1.4 Left panel Space filing model of the structure of bacterial dihydrofolate reductase with methotrexate bound to the active site. Right panel Close-up view of the active site, illustrating the structural complementarity between the ligand (methotrexate) and the binding pocket. See color insert. Source Courtesy of Nesya Nevins.

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... 

Bajorath, J., D. H. Kitson, G. Fitzgerald, J. Andzelm, J. Kraut, and A. T. Hagler. 1991. Local Density Functional Calculations on a Protein System Folate and Escherichia Coli dihydrofolate reductase. Electron Redistribution on Binding of a Substrate to an Enzyme. PROTEINS 9, 217. 

Drug efficacy is directly related to its intracellular concentration level, so it is necessary to evaluate the MTX concentration in cells. In particular, MTX is a folate antagonist, thus it binds to dihydrofolate reductase in competition with folate [71-77]. A low intracellular level of MTX caused by high efflux and low uptake in resistant cells is also the main disadvantage of MTX medication [78,79]. This leads to a high dosage of MTX for cancer treatment, which is also directly associated with adverse effects. 

Solid-phase synthesis of dihydropteridinones has been achieved from 4,6-dichloro-5-nitropyrimidine <00TL8177>. A series of ethyl 7-aminopteridine-6-carboxylate derivatives has been prepared in one step from the reaction of vicinal diamines as 13-dialky 1-5,6-diamino-2-thiouracils with diethyl ( )-2,3-dicyanobutenedioate <99JHC1317>. The relative binding affinities to human dihydrofolate reductase of new 2,4-diaminopteridine derivatives... 

The above model was used to study the binding of methotrexate, and analogues of it, to wild-type and mutant forms of human dihydrofolate reductase (DHFR).29 This turned out to be a particularly difficult test because of the three ionized groups of methotrexate. The overall electrostatic interactions of this inhibitor amount to around -500 kcal/mol and MD trajectories of length more than a ns were required in order to get average... 

Hagler, Structure and energetics of ligand binding to proteins Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system, Proteins 4 31 (1988). 

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