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Published on: March 18, 2012
Structural dynamics at the active site of the cancer-associated flavoenzyme NQO1 probed by chemical modification with
Alice Grieco1, Miguel A Ruiz-Fresneda2, Atanasio Gómez-Mulas3
1Department of Crystallography & Structural Biology, Institute of Physical Chemistry Blas Cabrera, Spanish National Research Council (CSIC), Madrid, Spain.
Abstract:
A large conformational heterogeneity of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with various human diseases, has been observed to occur in the catalytic site of the enzyme. Here, we report the X-ray structure of NQO1 with phenylmethylsulfonyl fluoride (PMSF) at 1.6 Å resolution. Activity assays confirmed that, despite being covalently bound to the Tyr128 residue at the catalytic site, PMSF did not abolish NQO1 activity. This may indicate that the PMSF molecule does not reduce the high flexibility of Tyr128, thus allowing NADH and DCPIP substrates to bind to the enzyme. Our results show that targeting Tyr128, a key residue in NQO1 function, with small covalently bound molecules could possibly not be a good drug discovery strategy to inhibit this enzyme.
Insights
Human NAD(P)H:quinone oxidoreductase 1 (NQO1) retains activity even when covalently bound to PMSF. This suggests targeting Tyr128 may not be an effective drug strategy for inhibiting NQO1.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein implicated in various diseases.
- Conformational heterogeneity in the NQO1 catalytic site is a known characteristic.
- Understanding NQO1's structural dynamics is crucial for therapeutic development.
Purpose of the Study:
- To determine the X-ray structure of NQO1 in complex with phenylmethylsulfonyl fluoride (PMSF).
- To investigate the impact of PMSF covalent binding on NQO1 activity and catalytic site flexibility.
- To evaluate the potential of targeting Tyr128 for NQO1 inhibition.
Main Methods:
- X-ray crystallography to obtain the NQO1-PMSF complex structure at 1.6 Å resolution.
- Enzyme activity assays to measure NQO1 function in the presence of PMSF.
Main Results:
- The crystal structure revealed PMSF covalently bound to the Tyr128 residue within the NQO1 catalytic site.
- Activity assays demonstrated that PMSF binding did not abolish NQO1 activity.
- The results suggest PMSF does not significantly restrict the flexibility of Tyr128, allowing substrate binding.
Conclusions:
- Covalent modification of Tyr128 with PMSF does not inhibit NQO1 activity.
- The flexibility of Tyr128 may be maintained despite PMSF binding, enabling substrate interaction.
- Targeting Tyr128 with small, covalently binding molecules may not be a viable strategy for developing NQO1 inhibitors.
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