ENGINEERING SPECIFICITY FOR FOLATE INTO DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI

Despite several similarities in structure and kinetic behavior, the ba cterial and vertebrate forms of the enzyme dihydrofolate reductase (DH FR) exhibit differential specificity for folate. In particular, avian DHFR is 400 times more specific for folate than the Escherichia coli r eductase. We proposed to enhance the specificity of the E. coli reduct ase for folate by incorporating discrete elements of vertebrate second ary structure. Two vertebrate loop mutants, VLI and VLII containing 3- 7 additional amino acid insertions, were constructed and characterized by using steady-state kinetics, spectrofluorimetric determination of ligand equilibrium dissociation constants, and circular dichroism spec troscopy. Remark ably, the VLI and VLII mutants are kinetically simila r to wild-type E. coli reductase when dihydrofolate is the substrate, although VLII exhibits prolonged kinetic hysteresis. Moreover, the VLI dihydrofolate reductase is the first mutant form of E. coli DHFR to d isplay enhanced specificity for folate [(k(cat)/K-m)(mutant)/ (k(cat)/ K-m)(wt) = 13]. A glycine-alanine loop (GAL) mutant was also construct ed to test the design principles for the VLI mutant. In this mutant of the VLI reductase, all of the residues from positions 50 to 60, excep t the strictly conserved amino acids Leu-57 and Arg-60, were converted to either glycine or alanine. A detailed kinetic comparison of the GA L and wild-type reductases revealed that the mutations weaken the bind ing by both cofactor and substrate by up to 20-fold, but under saturat ing conditions the enzyme exhibits a k(cat) value nearly identical to that of the wild type. The rate of hydride transfer is reduced by a fa ctor of 30, with a compensating increase in the dissociation rate for tetrahydrofolate. Although key stabilizing interactions have been sacr ificed (it shows no activity toward folate), the maintenance of the co rrect register between key residues preserves the activity of the enzy me toward its natural substrate. Collectively, neither specific proxim al point site mutations nor larger, more distal secondary structural s ubstitutions are sufficient to confer a specificity for folate reducti on that matches that observed with the avian enzyme. This is consisten t with the hypothesis that the entire protein structure must contribut e extensively to the enzyme's specificity.