ELECTROSTATIC CHARACTERIZATION OF ENZYME COMPLEXES - EVALUATION OF THE MECHANISM OF CATALYSIS OF DIHYDROFOLATE-REDUCTASE

The catalytic mechanism of dihydrofolate reductase is evaluated with P oisson-Boltzmann electrostatic and quantum chemical vibrational freque ncy calculations. The results indicate that an elevated pK(a) of 6.5 a ssociated with the chemical step is due to the formation of the enol t automer of the substrate's pterin ring. The tautomer is induced to for m as a result of substrate binding, in which the substrate desolvates the active site and binds to the carboxylate of Asp 27. Although the b inding reaction is favorable, burial of the negative charge on Asp 27 is not. Protonation of Asp 27 occurs, concerted with tautomerization o f the substrate, resulting in active site electrical neutrality and ac tivation of the substrate for catalysis. These results require a reint erpretation of previous data from Raman spectroscopy studies in which it was proposed that the reactive atom, the pterin N5, is directly pro tonated Quantum chemical vibrational frequency calculations demonstrat e that the enol tautomer undergoes a Raman active vibrational perturba tion at a frequency similar to that observed experimentally. Furthermo re, the calculations indicate that direct protonation of the pterin N5 due to classical electrostatic interactions is quite difficult, with the pK, for this residue being shifted from 2.6 in solution to below z ero while bound to the protein. The conclusions of this work are indep endent of the protein dielectric constant in the range of 4-20.