ROLE OF THE ACTIVE-SITE CARBOXYLATE IN DIHYDROFOLATE-REDUCTASE - KINETIC AND SPECTROSCOPIC STUDIES OF THE ASPARTATE 26-]ASPASAGINE MUTANT OF THE LACTOBACILLUS-CASEI ENZYME

A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in whic h the active site aspartic acid residue has been replaced by asparagin e by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differenc es between the methotrexate complexes of the wild-type and mutant enzy mes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of t he pteridine ring in the binding site in the mutant enzyme. Both NMR a nd UV spectroscopy show that methotrexate is protonated on N1 when bou nd to the wild-type enzyme but not when bound to the mutant. Binding c onstant measurements by fluorescence quenching and steady-state kineti c measurements of dihydrofolate (FH2) and folate reduction show that t he substitution has little or no effect on substrate, coenzyme, and in hibitor binding (<7-fold increase in K-d) and only a modest effect on k(cat) (up to a factor of 9 for FH2 and 25 for folate) and k(cat)/K-M (up to a factor of 13 for FH2 and 14 for folate). Measurements of deut erium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the muta nt enzyme hydride ion transfer is rate-limiting across the pH range 5- 8. This allowed a direct comparison of the rate of hydride ion transfe r in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5 . This effect is much smaller than that seen for the corresponding mut ant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villaf ranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hyd ride ion transfer. The change in pH dependence of k(cat) resulting fro m the substitution is consistent with, but does not prove, the idea th at the group of pK 6.0 which must be protonated for hydride ion transf er to occur is Asp26. For folate reduction, the pH dependence of k(cat ) is determined by two pKs, one of which, pK 5, disappears in the muta nt enzyme, suggesting that it may correspond to ionization of Asp26. T he second pK, 7.3 in both wild-type and 6.9 in the mutant enzyme, may be an ionization of the folate molecule. The small effect of the Asp26 --> Asn substitution in L. casei dihydrofolate reductase makes it unl ikely that this group acts as an essential proton donor in catalysis. The possible roles of Asp26 and Asn26 in catalysis are discussed.