DHFR is an enzyme that exists in all cells and plays an important role in the DNA replication. This enzyme catalyzes the hydride-transfer from NADPH to dihydrofolate (DHF). Also, this enzyme has monomeric, flexible, and small structure that makes it easy to study. This enzyme is biologicaly important as it accounts for the synthesis of DNA. The dihydrofolate reductase (DHFR) is essential for the metabolism of folic acid (vitamin B12). Folic acid is a crucial vitamin that accounts for the DNA synthesis.

We report the mechanistic study of the hydride reduction of 9,10-dimethylacridinium ion (DMA⁺) by 1-benzyl-1,4-dihydronicotinamide (BNAH) in acetonitrile. The reaction is a model for the enzymatic reactions in which hydride transfers between two carbons including the DHFR reactions. The BNAH is an NADPH model and the DMA⁺ is a model for DHFH⁺. The temperature dependence of 1° KIEs and the 2° KIEs at the positions of 9-CH ₃/CD₃ and 10-CH₃/CD₃ for both hydride and deuteride transfers were determined. The temperature over-dependence of 1° KIEs (A_H/A_D = 0.090), and the larger 2° KIE for H-transfer (1.07) than for D-transfer (0.98), strongly suggest H-tunneling. The 2° KIE on 10-methyl position is normal (1.02), consistent with the fact that the CD₃-NR₂ is a stronger base than CH ₃-NR₂. The results are explained by the Marcus-like H-tunneling model.