STRUCTURE OF DIHYDROFOLATE WHEN BOUND TO DIHYDROFOLATE-REDUCTASE

The Raman spectrum of dihydrofolate (H(2)folate)complexed with dihydro folate reductase (DHFR) and NADP(+), believed to be an accurate mimic of the productive DHFR/NADPH/H(2)folate complex involved in the reacti on catalyzed by DHFR, contains bands associated with stretch motions o f N5=C6 of bound substrate However, the assignments df these bands, wh ich are of considerable importance to understanding enzymic. mechanism and substrate binding, are in doubt. The vibrational spectra of dihyd rofolate, alone and complexed with water and with acetate, have been c alculated using quantum mechanical ab initio procedures in order to as sign the observed bands. Several structural conclusions follow from th ese calculations. N5 of H(2)folate when bound to DHFR/NADP(+) has a pK (a) of 6.5. From an examination of deuteration shifts, the immediate e nvironment of N5 of substrate is quite hydrophobic: there does not app ears to be an immediate water molecule near enough to form a hydrogen- bond with a protonated N5-H. It is suggested that the carboxyl group o f Asp27, the only ionizable group in the DHFR binding site, is ionized at physiological pH values and does not donate a proton to substrate during:enzymic catalysis. Overall, the results suggest that a major st ructural attribute of DHFR is to raise the pK(a) of N5 4 units when H( 2)folate binds in the productive ground-state ternary complex. Such a strategy would appear to be responsible for a substantial portion of t he rate enhancement in the reaction catalyzed by DHFR.