EFFECT OF ENZYME AND LIGAND PROTONATION ON THE BINDING OF FOLATES TO RECOMBINANT HUMAN DIHYDROFOLATE-REDUCTASE - IMPLICATIONS FOR THE EVOLUTION OF EUKARYOTIC ENZYME EFFICIENCY

There is marked pH dependence of the rate constant (k(off)) for tetrahydrofolate (H-4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H-4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31 --> Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH almost-equal-to 5, and this accounts for the marked decrease in k(cat) for this variant as the pH is decreased from 7 to 5. This decreased k(cat) at low pH is not seen for most DHFRs. k(off) for dissociation of folate, dihydrofolate (H-2folate), and H-4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of k(off) in the range pH 5-7 is well described by a pK(a) of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), k(off) increases further as the pH is raised, and this relation is governed by a second PK(a) which is close to the pK(a) for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases k(off). Evidence is presented that the dependence of pH on k(off) for hDHFR accounts for the shape of the k(cat) versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.