Direct measurement of the pK(a) of aspartic acid 26 in Lactobacillus caseidihydrofolate reductase: Implications for the catalytic mechanism

The ionization stale of aspartate 26 in Lactobacillus casei dihydrofolate r eductase has been investigated by selectively labeling the enzyme with [C-1 3 gamma] aspartic acid and measuring the C-13 chemical shifts in the ape, f olate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pK(a) greater than similar to 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolat e-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pK(a) value of less than 4 in this complex. Such a lo w pK(a) value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pK(a) = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-575 0]. Furthermore, the downfield chemical shift of the Asp 26 C-13 gamma reso nance in the dihydrofolate-enzyme complex provides experimental evidence th at the pteridine ring of dihydrofolate is polarized when bound to the enzym e. We propose that this polarization of dihydrofolate acts as the driving f orce for protonation of the electron-rich O4 atom which occurs in the prese nce of NADPH. After this protonation of the substrate, a network of hydroge n bonds between O4, N5 and a bound water molecule facilitates transfer of t he proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.