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Understanding substrate specificity in human and parasite phosphoribosyltransferases through calculation and
J W Pitera1, N R Munagala, C C Wang
1Graduate Group in Biophysics and Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, San Francisco, California 94143-0446, USA.
Biochemistry
|August 11, 1999
Summary
Molecular dynamics simulations reveal that a more flexible base-binding region in the parasite HGXPRTase explains its broader substrate specificity compared to human HGPRTase. This study also demonstrates the utility of computational tools for designing species-selective inhibitors.
Area of Science:
- Biochemistry
- Computational Biology
- Enzymology
Background:
- Human hypoxanthine-guanine-phosphoribosyltransferase (HGPRTase) is crucial for purine salvage.
- The protozoan parasite Tritrichomonas foetus possesses a related enzyme, HGXPRTase, with broader substrate specificity, including xanthine.
- Subtle active site differences exist between human HGPRTase and T. foetus HGXPRTase.
Purpose of the Study:
- To investigate the molecular basis for the broader substrate specificity of T. foetus HGXPRTase using molecular dynamics (MD) simulations.
- To rationalize the effects of specific mutations on enzyme substrate specificity.
- To demonstrate the utility of computational methods for designing species-selective enzyme inhibitors.
Main Methods:
- Molecular dynamics (MD) simulations of human HGPRTase and T. foetus HGXPRTase.
- Analysis of enzyme active site flexibility and substrate-binding interactions.
- Computational design of enzyme inhibitors using the PROFEC program.
- Experimental validation of a predicted double mutant enzyme.
Main Results:
- MD simulations indicated a significantly more flexible base-binding region in the parasite HGXPRTase, explaining its broader substrate specificity.
- The study rationalized the substrate specificity alterations caused by mutations R155E and D163N.
- A double mutant (D106E/D163N) was computationally predicted to rescue the D163N mutation and was experimentally confirmed.
- Computational design using PROFEC suggested parasite-selective GMP derivatives and rationalized existing novel inhibitors.
Conclusions:
- The broader specificity of T. foetus HGXPRTase is attributed to increased flexibility in its base-binding region.
- Computational simulations and structure-based design are powerful tools for understanding enzyme mechanisms and developing species-selective inhibitors.
- The PROFEC program is effective for designing novel enzyme inhibitors with targeted selectivity.