95790 20210902121925.0 doi 10.3390/antiox9090772 sideral 120225 ART-2020-120225 eng Anoz-Carbonell, E. Universidad de Zaragoza (orcid)0000-0002-6649-9153 The catalytic cycle of the antioxidant and cancer-associated human NQO1 enzyme: Hydride transfer, conformational dynamics and functional cooperativity 2020 Access copy available to the general public Unrestricted 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. info:eu-repo/grantAgreement/ES/DGA-FEDER/E35-20R info:eu-repo/grantAgreement/ES/MCIU-ERDF/RTI2018-096246-B-I00 info:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-103901GB-I00 info:eu-repo/semantics/openAccess by http://creativecommons.org/licenses/by/3.0/es/ 6.312 2020 BIOCHEMISTRY & MOLECULAR BIOLOGY 60 / 297 = 0.202 2020 Q1 T1 FOOD SCIENCE & TECHNOLOGY 11 / 144 = 0.076 2020 Q1 T1 CHEMISTRY, MEDICINAL 6 / 63 = 0.095 2020 Q1 T1 1.066 2020 Biochemistry 2020 Q2 Cell Biology 2020 Q2 Physiology 2020 Q2 Molecular Biology 2020 Q2 Clinical Biochemistry 2020 Q2 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Timson, D.J. Pey, A.L. Medina, M. Universidad de Zaragoza (orcid)0000-0001-8743-0182 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 9, 9 (2020), 772 [1-22] Antioxidants Antioxidants 2076-3921 980970 http://zaguan.unizar.es/record/95790/files/texto_completo.pdf Versión publicada 476236 http://zaguan.unizar.es/record/95790/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:95790 articulos driver 2021-09-02-10:50:55 ARTICLE