000170380 001__ 170380
000170380 005__ 20260420103354.0
000170380 0247_ $$2doi$$a10.1111/febs.70511
000170380 0248_ $$2sideral$$a148927
000170380 037__ $$aART-2026-148927
000170380 041__ $$aeng
000170380 100__ $$aRivero, Maribel
000170380 245__ $$aTyrosine residues at the substrate binding site in human NQO1 homodimer: Protein conformational dynamics and optimization of substrate binding geometry
000170380 260__ $$c2026
000170380 5060_ $$aAccess copy available to the general public$$fUnrestricted
000170380 5203_ $$aHuman NQO1 is a homodimeric flavoenzyme essential for the redox metabolism of many substances and implicated in major global health challenges such as cancer and Alzheimer's disease. X-ray crystallographic studies have identified several residues within its substrate binding site (including Tyr126 and Tyr128) that may regulate catalytic competent binding of substrates, cofactor redox properties, half-site reactivity, and/or functional inter-active site negative cooperativity. To elucidate the functional role of Tyr126 and Tyr128, we generated point mutants at these positions and assessed their dynamics and kinetic properties. Hydrogen-deuterium exchange coupled to mass spectrometry revealed that non-conservative mutations, particularly at Tyr126, notably disrupted dynamics not only within the substrate binding site but also in structural elements connecting the two active sites of the NQO1 homodimer. Rapid-mixing pre-steady-state kinetics experiments of the reduction of NQO1 by NAD(P)H showed that mutations to Phe caused a mild decrease in hydride transfer (HT) efficiency from the coenzyme to the FAD cofactor. In contrast, mutations to Ala resulted in a significantly greater impact and mutations to Glu nearly abolished HT. Despite these effects, some mutations moderately affected the non-synchronous catalysis between the two alternating active sites, but hardly produced an impact on the selectivity for NADPH versus NADH as hydride donor coenzymes. However, all variants exhibited markedly impaired enzyme turnover, highlighting alterations in the enzyme's substrate specificity toward quinones. The data presented here demonstrate that Tyr126 and Tyr128 optimize both substrate binding geometry as well as overall enzyme conformational dynamics during the asymmetric catalytic cycle of the NQO1 homodimer.
000170380 536__ $$9info:eu-repo/grantAgreement/ES/DGA-FEDER/E35-23R$$9info:eu-repo/grantAgreement/ES/MICINN/PID2022-136369NB-I00
000170380 540__ $$9info:eu-repo/semantics/openAccess$$aby-nc-nd$$uhttps://creativecommons.org/licenses/by-nc-nd/4.0/deed.es
000170380 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000170380 700__ $$aPacheco-Garcia, Juan Luis
000170380 700__ $$aVankova, Pavla
000170380 700__ $$aLoginov, Dmitry
000170380 700__ $$aQuereda-Moraleda, Isabel
000170380 700__ $$aMartin-Garcia, Jose Manuel
000170380 700__ $$aMan, Petr
000170380 700__ $$aPey, Angel Luis
000170380 700__ $$0(orcid)0000-0001-8743-0182$$aMedina, Milagros$$uUniversidad de Zaragoza
000170380 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole.
000170380 773__ $$g(2026), [27 pp.]$$pFEBS J.$$tFEBS Journal$$x1742-464X
000170380 8564_ $$s4399366$$uhttps://zaguan.unizar.es/record/170380/files/texto_completo.pdf$$yVersión publicada
000170380 8564_ $$s2537655$$uhttps://zaguan.unizar.es/record/170380/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000170380 909CO $$ooai:zaguan.unizar.es:170380$$particulos$$pdriver
000170380 951__ $$a2026-04-18-10:48:25
000170380 980__ $$aARTICLE