Reversal of Methotrexate Binding to Dihydrofolate Reductase by Dihydrofolate STUDIES WITH PURE ENZYME AND COMPUTER MODELING USING NETWORK THERMODYNAMICS* (Received for publication, June 1, 1979)

The folate analog, methotrexate, binds very tightly to dihydrofolate reductase. However, free intracellular methotrexate in excess of the enzyme-binding capacity is needed to suppress cellular dihydrofolate reduction, and the effects of methotrexate are rapidly reversible if methotrexate is removed from the extracellular media. The apparent paradox may be explained on the basis of a single high affinity methotrexate binding site if the competitive effect of the substrate, dihydrofolate, on inhibitor binding is taken into consideration. At concentrations above the K,,,, dihydrofolate can inhibit [3H]methotrexate binding to a homogeneous preparation of dihydrofolate reductase from Escherichia coli MB 1428. The effects of dihydrofolate on methotrexate binding at 37°C in the presence of excess NADPH are time-dependent since substrate binding results in enzymatic conversion to tetrahydrofolate thus reducing the dihydrofolate concentration. Incubation of the [3H]methotrexate l dihydrofolate reductase l NADPH ternary complex with dihydrofolate results initially in displacement of [3H]methotrexate similar to that induced by unlabeled methotrexate. As the dihydrofolate concentration is depleted, competition with methotrexate for binding sites is relieved and ultimately all displaced [3H]methotrexate reassociates with the enzyme. Using rate constants determined experimentally or derived from literature values, including methotrexate Ki = 5 x lo-l2 M, this experiment was simulated by a computer method based on network thermodynamics. The computer simulation closely reproduces the experimentally determined results indicating strongly that these observations reflect interactions among substrate, inhibitor, and enzyme at the high affinity methotrexate binding site. The computer model was extended to include membrane transport and thymidylate synthetase activity to reflect occurrences within a cell more closely. The computer analysis indicates that when free methotrexate is low but intracellular dihydrofolate is high, displacement of bound methotrexate by dihydrofolate plays an important role in reversing the toxic effects of this agent.