Dihydrofolate reductase active site structure

Fig. 5. Active-site structure of dihydrofolate reductase, showing selected amino acids involved in substrate binding. [Redrawn combining information from Refs. (70), (136), and (/5J).)...

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.

Aminopterin and methotrexate are anticancer drugs that are inhibitors of dihydrofolate reductase. Because their structures are similar to that of dihydrofolate, they compete with it for binding to the active site of the enzyme. Because they hind 1000 times more tightly to the enzyme than dihydrofolate does, they win the competition and thwefore inhibit the enzyme s activity. These two compounds are examples of competitive inhibitors. 

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.

Figure 15-19 Drawings of the active site of E. coli dihydrofolate reductase showing the hound ligands NADP+ and tetrahydrofolate. Several key amino acid side chains are shown in the stereoscopic views on the right. The complete ribbon structures are on the left. (A) Closed form. (B) Open form into which substrates can enter and products can escape. From Sawaya and Kraut.381 Courtesy of Joseph Kraut. Molscript drawings (Kraulis, 1991).

Some of the structural properties which have been ascribed to DA receptors appear to deserve attention for their heuristic value, but painfully few should engender much confidence in their reality. A sobering lesson is available from analysis of complexes of dihydrofolate reductase (], 8). Methotrexate is a very close analog of folic acid and is a potent inhibitor of the enzyme, but it is now almost certain that these ligands bind in the enzyme active site in aspects differing by a rota-... 

Another enzyme for which X-ray diffraction studies have aided in an analysis of the mode of action is the enzyme dihydrofolate reductase. This catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential coenzyme used in the synthesis of thymidylate, inosinate, and methionine. The antitumor agent methotrexate is a powerful inhibitor of dihydrofolate reductase, causing, on binding, a cellular deficiency of thymidylate (the cause of its antitumor activity). The crystal structures of the enzyme from two bacterial sources—Escherichia coli and Lactobacillus casei—and from chicken liver have been studied (88-90). Both the E. coli and L casei enzymes have been studied as complexes with methotrexate bound at the active site, and, in the case of the . casei enzyme, the cofactor, NADPH, was also present. 

Another characteristic of competitive inhibition is structural similarity between substrate and competitive inhibitor. The similarity allows the inhibitor to bind in the active site of the enzyme. For example, the enzyme dihydrofolate reductase (DHFR) is subject to competitive inhibition by methotrexate and other compounds. Methotrexate is used in cancer chemotherapy because DHFR is required for the synthesis of thymidine triphosphate, a specific precursor of DNA (Fig. 8.8). The normal substrate for DHFR is folate and methotrexate is closely related (Fig. 8.2). 

The active-site cavities of the murine, avian, E. coli, and L. casei dihydrofolate reductases show similar surface contours, yet the bacterial enzymes only slowly reduce folate and are inhibited to a greater extent by trimethoprim (148). One notable structural difference is the greater length of an internal loop element in the avian and murine dihydrofolate reductases in a sequence spanning residues 51 to 59 (in E. coli) and connecting the C a helix and fi sheet that flank the aromatic side chain of the ligands. The influence of this loop on the enzymic... 

Figure 52 Interaction potential of an amino group with the active site of Escherichia coli dihydrofolate reductase, calculated by GRID the water molecule H39 is treated as a structural part of the enzyme in the calculations. The energy contours are plotted at — 63 kj mol they indicate two sites of strong attraction. The position of trimethoprim relative to the active site was adjusted so that the amino nitrogens N2 and N4 are located in these regions (reproduced from Figure 8 of ref. [33] with permission from the American Chemical Society, Washington. DC, USA).

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