PXD060224-1

PXD060224 is an original dataset announced via ProteomeXchange.

Title	Active site tyrosine residues in human NQO1 homodimer are critical for non-synchronous enzyme catalysis at the two active sites
Description	Human NQO1 is a flavoenzyme essential for the redox metabolization of many substances and associated with wide-impacting diseases such as cancer and Alzheimer´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 to Glu virtually abolished NQO1 activity. Interestingly, structural stability studies by HDX-MS showed significant perturbations that affected 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 and suggests that phosphorylation of these two Tyr residues might shut down NQO1 activity reversibly.
HostingRepository	PRIDE
AnnounceDate	2026-03-25
AnnouncementXML	Submission_2026-03-25_07:16:05.707.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dmitry Loginov
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	No PTMs are included in the dataset
Instrument	timsTOF Pro

Revision	Datetime	Status	ChangeLog Entry
0	2025-01-27 01:21:55	ID requested
⏵ 1	2026-03-25 07:16:06	announced

10.1111/FEBS.70511;

submitter keyword: enzyme kinetics, flavin, quinone oxidoreductase,Hydride transfer, coenzyme specificity

Petr Man
contact affiliation	Institute of Microbiology,The Czech Academy of Sciences
contact email	pman@biomed.cas.cz
lab head
Dmitry Loginov
contact affiliation	BioCeV - Institute of Microbiology of the CAS
contact email	dloginov@jcu.cz
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD060224

PRIDE project URI

• PRIDE
  1. PXD060224
     1. Label: PRIDE project
     2. Name: Active site tyrosine residues in human NQO1 homodimer are critical for non-synchronous enzyme catalysis at the two active sites