John A. Montgomery

Protonation of methotrexate bound to the catalytic site of dihydrofolate reductase from lactobacillus casei

Summary [2- 13 C]Methotrexate bound to dihydrofolate reductase from Lactobacillus casei has been studied by nuclear magnetic resonance. The chemical shift for enzyme-bound methotrexate over the range pH 6.0 to 9.0 is constant and close to that for free, protonated methotrexate. Furthermore, the ultraviolet spectrum of bound methotrexate is the same from pH 6.4 to pH 10.0 and considerably different from that of free, unprotonated methotrexate. These data indicate that N-1 of bound methotrexate has pK > 10. From this and from the measured apparent association constants of methotrexate and folate it can be concluded that the charge interaction between the protonated N-1 of bound methotrexate and the active site carboxyl accounts for the particularly tight binding of the inhibitor.