Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate

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Abstract
The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined. The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps. The V and V/KNADPH profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the Ki profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent. From the results of deuterium isotope effects on V/KNAPDH, it is concluded that NADPH behaves as a sticky substrate. It is this stickiness that raises artifically the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex [Howell, E.E., Villafranca, J.E., Warren, M. S., Oatley, S.J., and Kraut,J. (1986) Science (Washington, D.C.) 231, 1123] to an observed value of 8.9. Thus, the binary enzyme complex is largely protonated at neutral pH. The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates. Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step. At neutral pH, the proportion of enzyme present as a protonated ternary enzyme-substrate complex is sufficient to keep catalysis faster than prouct release. The data for deuterium isotope and deuterium solvent isotope effects are consistent with the postulate that, for the reduction of dihydrofolate to tetrahydrofolate, protonation precedes hydride transfer. A scheme is proposed for the indirect transfer of a proton from the enzyme to dihydrofolate. Dihydroflate reductase is another enzyme whose catalytic efficiency is limited by product release rather than by the chemistry of the reaction.

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