期刊
ANTIOXIDANTS
卷 9, 期 9, 页码 -出版社
MDPI
DOI: 10.3390/antiox9090772
关键词
antioxidant enzyme; antioxidant response; cancer; oxidoreductase; enzyme kinetic analysis; functional cooperativity; hydride transfer; kinetic isotope effects; quantum tunneling; conformational dynamics
资金
- ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency [RTI2018-096246-B-I00]
- Spanish Ministry of Science and Innovation-State Research Agency [PID2019-103901GB-I00]
- Consejeria de Economia, Conocimiento, Empresas y Universidad, Junta de Andalucia [P11-CTS-07187, P18-RT-2413]
- Gobierno de Aragon-FEDER [E35_20R]
Human NQO1 [NAD(H):quinone oxidoreductase 1] is a multi-functional and stress-inducible dimeric protein involved in the antioxidant defense, the activation of cancer prodrugs and the stabilization of oncosuppressors. Despite its roles in human diseases, such as cancer and neurological disorders, a detailed characterization of its enzymatic cycle is still lacking. In this work, we provide a comprehensive analysis of the NQO1 catalytic cycle using rapid mixing techniques, including multiwavelength and spectral deconvolution studies, kinetic modeling and temperature-dependent kinetic isotope effects (KIEs). Our results systematically support the existence of two pathways for hydride transfer throughout the NQO1 catalytic cycle, likely reflecting that the two active sites in the dimer catalyze two-electron reduction with different rates, consistent with the cooperative binding of inhibitors such as dicoumarol. This negative cooperativity in NQO1 redox activity represents a sort of half-of-sites activity. Analysis of KIEs and their temperature dependence also show significantly different contributions from quantum tunneling, structural dynamics and reorganizations to catalysis at the two active sites. Our work will improve our understanding of the effects of cancer-associated single amino acid variants and post-translational modifications in this protein of high relevance in cancer progression and treatment.
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