Active site-directed double mutants of dihydrofolate reductase Journal Article
| Authors: | Ercikan-Abali, E. A.; Mineishi, S.; Tong, Y.; Nakahara, S.; Waltham, M. C.; Banerjee, D.; Chen, W.; Sadelain, M.; Bertino, J. R. |
| Article Title: | Active site-directed double mutants of dihydrofolate reductase |
| Abstract: | Variants of dihydrofolate reductase (DHFR), which confer resistance to antifulates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22- Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active- site residues. Substrate and antifolate binding properties of these 'double' mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified fur one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells. |
| Keywords: | methotrexate; polymerase chain reaction; animals; mice; amino acid substitution; enzyme activity; structure activity relation; tyrosine; gene transfer; cancer resistance; kinetics; recombinant proteins; substrate specificity; gene therapy; mutagenesis, site-directed; binding sites; catalysis; point mutation; dihydrofolate reductase; folic acid antagonist; folic acid antagonists; tetrahydrofolate dehydrogenase; site directed mutagenesis; drug binding; phenylalanine; 3t3 cells; variation (genetics); humans; human; priority journal; article |
| Journal Title: | Cancer Research |
| Volume: | 56 |
| Issue: | 18 |
| ISSN: | 0008-5472 |
| Publisher: | American Association for Cancer Research |
| Date Published: | 1996-09-15 |
| Start Page: | 4142 |
| End Page: | 4145 |
| Language: | English |
| PUBMED: | 8797582 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 22 November 2017 -- Source: Scopus |
