Ionization state and pKa of pterin-analogue ligands bound to dihydrofolate reductase

The ionization state and pKa of the inhibitor 6,8-dimethyl-N5-deazapterin bound to the recombinant human dihydrofolate reductase (rhH2folate reductase) complex with NADPH was determined by a spectrofluorimetric method. The excitation spectra for bound ligand as a function of pH from 6.1 to 9.7 indicated it was the same cationic form as for unbound ligand, which is protonated on N3. However, the lower limit for the pKa of the bound form was determined to be 9.1, a value about pH 2.5 higher than that for free ligand, indicating that ligand bound to the enzyme is protonated at neutral pH. The excitation spectra for bound ligand as a function of pH were generated by computer simulation by employing corrections for the pH dependence of the proportion of bound ligand (variable Kd; ligand-dissociation constant) and taking account of the different pKa values for bound and unbound forms. A plot of Kd values against pH showed a bell-shaped curve indicating that 6,8-dimethyl-N5-deazapterin bound to rhH2folate reductase.NADPH to form a ternary complex of ionised enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand; the spectrofluorimetric results are consistent with the first alternative.