Active site tyrosine residues in human NQO1 homodimer are critical for non-synchronous enzyme catalysis at the two active sites.

Rivero, M.; Pacheco-Garcia, J. L.; Vankova, P.; Loginov, D. S.; Quereda-Moraleda, I.; Martin-Garcia, J. M.; Man, P. M.; Medina, M.; Pey, A. l.

doi:10.1101/2025.02.02.636097

bioRxiv · 10.1101/2025.02.02.636097

Active site tyrosine residues in human NQO1 homodimer are critical for non-synchronous enzyme catalysis at the two active sites.

Rivero, M.·Pacheco-Garcia, J. L.·Vankova, P.·Loginov, D. S.·Quereda-Moraleda, I.·Martin-Garcia, J. M.·Man, P. M.·Medina, M.·Pey, A. l.

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Abstract

AbstractHuman NQO1 is a flavoenzyme essential for the redox metabolization of many substances and associated with wide-impacting diseases such as cancer and Alzheime[r]s. Recent X-ray crystallographic studies have proposed that a few residues at the active site of NQO1 (including Tyr126 and Tyr128) may control enzyme catalysis and functional negative cooperativity. In this work, we use rapid mixing pre-steady state kinetics and hydrogen-deuterium exchange followed by mass spectrometry (HDX-MS) to evaluate experimentally the role of Tyr126 and Tyr128 in NQO1 functionality by generating mutants to Phe, Ala and Glu. Mutations to Phe caused mild effects, whereas those to Ala significantly decreased hydride transfer efficiency and those to Glu virtually abolished NQO1 activity. Interestingly, structural stability studies by HDX-MS showed significant perturbations particularly affecting the binding site of NADH/NAD+ in the less conservative mutations (particularly to Glu). Mutations of Tyr126 and Tyr128 seem to also modulate the non-synchronous catalysis in the two active sites (negative cooperativity) as well as the selectivity for NADH/NADPH as coenzymes. Our work experimentally demonstrates the critical role of Tyr126 and Tyr128 in the flavin reductive half-reaction of the catalytic cycle of NQO1 in the negative cooperativity, and also suggests that phosphorylation of these two Tyr residues might shut down NQO1 activity reversibly.

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Rivero, M., Pacheco-Garcia, J. L., Vankova, P., Loginov, D. S., Quereda-Moraleda, I., Martin-Garcia, J. M., Man, P. M., Medina, M., Pey, A. l.. 2025-02-07. Active site tyrosine residues in human NQO1 homodimer are critical for non-synchronous enzyme catalysis at the two active sites.. https://doi.org/10.1101/2025.02.02.636097

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