Dihydrofolate Reductase from the Pathogenic Fungus Pneumocystis carinii: Catalytic Properties and Interaction with Antifolates

• Published in: Archives of biochemistry and biophysics Vol. 305; no. 2; pp. 499 - 508
• Main Authors: Margosiak, S.A., Appleman, J.R., Santi, D.V., Blakley, R.L.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.09.1993; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Escherichia coli - enzymology; Folic Acid Antagonists - pharmacology; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen - chemistry; Hydrogen-Ion Concentration; Kinetics; Oxidoreductases; Pneumocystis - enzymology; Pneumocystis carinii; Protein Binding; Tetrahydrofolate Dehydrogenase - metabolism; Human; Protozoa; Enzyme; Escherichia coli; Molecular interaction; Pneumocystis carinii; Fungi; Enzymatic activity; Bacteria; Kinetics; Sporozoa; Comparative study; Enterobacteriaceae; Thallophyta; Reductase
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1993.1453

Dihydrofolate reductase (DHFR) from the fungus Pneumocystis carinii (pcDHFR), a target for antifolate inhibitors, has been compared with host enzyme, human DHFR (hDHFR), and with DHFR from Escherichia coli. Among the results of the considerable structural differences between pcDHFR and the other two enzymes is a much higher turnover number ( k cat, 136 s −1) for pcDHFR. This is due to rapid hydride transfer from NADPH to dihydrofolate (rate constant 402 s −1), very rapid dissociation of NADP from the product complex (rate constant, k off, > 1000 s −1), and after NADPH binding, rapid dissociation of tetrahydrofolate ( k off, 216 s −1). Cycling of pcDHFR is almost exclusively by this pathway. The high k cat contributes to a high K m for NADPH (9 μM) and an unusually high K m for dihydrofolate (2.5 μM). Nevertheless, the efficiency of pcDHFR is greater than DHFR from E. coli and about 25% that of hDHFR. Of seven clinically relevant inhibitors investigated, only one (trimethoprim) had a slightly lower K i for pcDHFR than for hDHFR. The therapeutic value of trimethoprim-sulfa treatment of P. carinii infections indicates that other factors play an important role, but the results are consistent with the frequency of complications due to toxicity of trimethoprim.