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Drug selectivity is determined by coupling across the NAD+ site of IMP dehydrogenase
1Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Abstract:
Drug resistance often results from mutations that are located far from the drug-binding site. The effects of these mutations are perplexing. The inhibition of IMPDH by MPA is an example of this phenomenon. Mycophenolic acid (MPA) is a species-specific inhibitor of IMPDH; mammalian IMPDHs are very sensitive to MPA, while the microbial enzymes are resistant to the inhibitor. MPA traps the covalent intermediate E-XMP and binds in the nicotinamide half of the dinucleotide site. Previous results indicated that about half of the difference in sensitivity derives from residues in the MPA-binding site [Digits, J. A., and Hedstrom, L. (1999) Biochemistry 38, 15388-15397]. The remainder must be attributed to regions outside the MPA-binding site. The adenosine subsite of the NAD+ site is not conserved among IMPDHs and is, therefore, a likely candidate. Our goal is to examine the coupling between the nicotinamide and adenosine sites in order to test this hypothesis. We performed multiple inhibitor experiments with the Tritrichomonas foetus and human type 2 IMPDHs using tiazofurin and ADP, which bind in the nicotinamide and adenosine subsites, respectively. For T. foetus IMPDH, tiazofurin and ADP are extraordinarily synergistic. In contrast, these inhibitors are virtually independent for the human type 2 enzyme. We suggest that the difference in coupling of the nicotinamide and adenosine subsites accounts for the remaining difference in MPA affinity between T. foetus and human IMPDH.
Insights
Drug resistance in inosine monophosphate dehydrogenase (IMPDH) can arise from distant mutations. Differences in enzyme-inhibitor coupling between human and microbial IMPDH explain varying sensitivity to mycophenolic acid (MPA).
Area of Science:
- Biochemistry
- Enzyme kinetics
- Drug resistance mechanisms
Background:
- Drug resistance frequently stems from mutations distant from the drug-binding site, a perplexing phenomenon.
- Mycophenolic acid (MPA) is a species-specific inhibitor of inosine monophosphate dehydrogenase (IMPDH), with high sensitivity in mammalian enzymes and resistance in microbial counterparts.
- MPA binds to the nicotinamide half of the dinucleotide site, trapping a covalent intermediate. Previous studies attributed half the sensitivity difference to residues within the MPA-binding site.
Purpose of the Study:
- To investigate the coupling between the nicotinamide and adenosine sites of IMPDH to explain drug resistance.
- To test the hypothesis that the adenosine subsite, which is not conserved among IMPDHs, contributes to species-specific drug sensitivity.
Main Methods:
- Performed multiple inhibitor experiments using Tritrichomonas foetus (microbial) and human type 2 IMPDH.
- Utilized tiazofurin, which binds to the nicotinamide subsite, and ADP, which binds to the adenosine subsite.
- Assessed the synergistic or independent interactions of tiazofurin and ADP binding in both enzyme types.
Main Results:
- Tiazofurin and ADP exhibited extraordinary synergy when used with T. foetus IMPDH.
- In contrast, tiazofurin and ADP showed virtually independent binding for human type 2 IMPDH.
- These findings indicate significant differences in the coupling between the nicotinamide and adenosine subsites.
Conclusions:
- The differential coupling between the nicotinamide and adenosine subsites in IMPDH is proposed to account for the remaining differences in MPA sensitivity between species.
- This study elucidates a key mechanism underlying species-specific drug inhibition and resistance in IMPDH.