Dihydrofolate reductase substrate interactions

Figure 1.5 Interactions of the dihydrofolate reductase active site with the inhibitor methotrexate (left) and the substrate dihydrofolate (right).

P.H. Liang and K.S. Anderson. 1998. Substrate channeling and domain-domain interactions in bifunctional thymidylate synthase-dihydrofolate reductase Biochemistry 37 12195-12205. (PubMed)... 

To illustrate the relevance of pharmacophore centers. Fig. 10 shows two ways of superimposing a dihydrofolate and a methotrexate molecule. Both compounds bind to the enzyme dihydrofolate reductase. Dihydrofolate is the natural substrate, methotrexate is a potent inhibitor. Atom-by-atom mapping leads to the first superposition. By looking at pharmacophore centers and the direction of interactions a quite different superposition, in which a part of the molecule is rotated by 180°, is found. Indeed, this is the orientation found by X-ray analyses of the enzyme-ligand complexes [73]. 

FIGURE 6-4 Complementary shapes of a substrate and its binding site on an enzyme. The enzyme dihydrofolate reductase with its substrate NADP" (red), unbound (top) and bound (bottom). Another bound substrate, tetrahydrofolate (yellow), is also visible (PDB ID 1 RA2).The NADP binds to a pocket that is complementary to it in shape and ionic properties. In reality, the complementarity between protein and ligand (in this case substrate) is rarely perfect, as we saw in Chapter 5. The interaction of a protein with a ligand often involves changes in the conformation of one or both molecules, a process called induced fit. This lack of perfect complementarity between enzyme and substrate (not evident in this figure) is important to enzymatic catalysis. 

Scheme I. The preferred pathway is represented by the closed loop (heavier arrows), which bypasses free enzyme. Following the release of NADP+, the release of H4F from the E-H4F complex is too slow to account for turnover, but it is enhanced 10-fold by the binding of NADPH at the neighboring site. Studies on dihydrofolate reductase have provided a complete analysis of the effects of point mutations on the rate and equilibrium constants for each step in the reaction sequence. The active site structure of dihydrofolate reductase is shown schematically in Fig. 5, illustrating some of the amino acids interacting with the substrates that have been mutated.

Selected examples of apparent free energies of interaction attributable to single amino acid side chains are summarized for three enzymes, T4 lysozyme, dihydrofolate reductase (DHFR), and tyrosyl-tRNA synthetase (TRS) (see Figs. 1,5, and 12, respectively, for structural information). In each case, the thermodynamic effect of the substitution was quantitated in terms of reduced binding affinity for the substrate or transition state, or reduced thermostability in the case of T4 lysozyme. 

The effect of the electrostatic interactions on the catalytic mechanism of dihydrofolate reductase (DHFR) from E. coli has been undertaken by Cannon et al.130 The study of two enzyme-cofactor complexes and two enzyme-cofactor-substrate complexes shows that Asp-27 plays a major role in catalysis, and that the formation of 4-hydroxypterin is the more likely intermediate than... 

An extensively studied enzyme-inhibitor system involves the protein dihydrofolate reductase [88]. Crystallographic results demonstrate an important feature of drug-enzyme interactions an inhibitor drug may not bind in the same way as substrate even though both have similar chemical formulae. This enzyme catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential... 

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