We continue our studies on dihydrofolate reductase (DHFR), a key enzyme in folic acid metabolism and the target site for a series of important drugs that are structural analogs of the vitamin, folic acid. Although the liver is the major site for this reductase activity, initial attempts to investigate DHFR in human liver was deterred by the observation that enzyme activity was extremely low in this tissue. However, the human reductase (hrDHFR) has now been cloned and expressed in E. coli by Dr. James Freisheim, Medical College of Ohio and is available in somewhat large amounts. In a continuing collaboration, I am examining certain activation phenomenon and the properties of a specific sulfhydryl group in the human enzyme such as I previously discovered and studied in DHFR derived from the chick, beef, pork, and sheep. In studies, thus far, the hrDHFR responds to -SH modification in a manner similar to that observed with the beef reductase. About 2-fold activation is noted after treatment with p-chloromercuribenzoate (pcmb). Other organic mercurials exhibit neither activation nor inhibition. This is to be compared with the chicken liver reductase (clDHFR) that is activated by all organic mercurials examined to date. In addition, tetrathionate and iodine that activate clDHFR as much as 5-fold have no effect on the hrDHFR. Again, the maximum transient activation by 4-5 M urea is only about 2-fold, whereas the same urea concentration yields about 5-fold with the clDHFR. Whether this is the maximum activation that can be induced in the human enzyme as compared with the dramatic increases in activity observed with the clDHFR is the basis for current studies. The possibilities of relating these observations to differences in the structure of the various DHFRs, particularly in the region of the-SH that is removed from the active site are being explored. In addition, a hypothesis relating these observations to a potential physiological function is under consideration.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
United States
Zip Code