The mechanism of binding of folate analogues by folate reductase.

The first step in the conversion of folate to the functional form of the vitamin is its reduction to a tetrahydro derivative (1). The enzyme, folate reductase (also called dihydrofolate reductase or tetrahydrofolate dehydrogenase), which catalyzes this reaction has been partially purified from a variety of tissues in different laboratories (2-8). Aminopterin and amethopterin are potent inhibitors of this enzyme (2, 4, 5, 8-10). Werkheiser (11) demonstrated that both inhibitors are bound to the reductase so tightly that, from the biological point of view, the inhibition appears to be irreversible. Since at very high excess of substrate over the inhibitor, the drug could be displaced from the ens”yme, the term “stoichiometric” rather than “irreversible” was suggested for this type of inhibition (11). It was estimated that aminopterin is bound to the reductase approximately 100,000 times more tight,ly than folate (11). It was suggested that the difference in the affinities to the reductase between folate and aminopterin is due to the stronger basicity of the latter compound (12, 13). The purpose of the present investigation was to determine the mechanism of binding to folate reductase of folate as compared to that of aminopterin. The effect on folic acid reductase of model compounds (pteridines, purines, and pyrimidines), some of them representing fragments of the molecules of folate and aminopterin, was studied. None of the various compounds resembling the 2-amino4hydroxypteridine ring of folate was capable of forming a complex with the enzyme. In the case of compounds resembling aminopterin, however, the formation of a complex with the enzyme could be demonstrated even when the molecules represented only the pyridimine portion, provided that the diamino structure was preserved. The thermodynamics of the binding of these compounds to the enzyme were also investigated. A new theory on the mechanism of binding of aminopterin and folate to the reductase which resulted from these studies will be discussed. Preliminary reports on this work have been presented (14, 15).