β-Carotene alleviates substrate inhibition caused by asymmetric cooperativity

Enzymes are essential catalysts in biological systems. Substrate inhibition, once dismissed, is now observed in 20% of enzymes¹ and is attributed to the formation of an unproductive enzyme-substrate complex, with no structural evidence of unproductivity provided to date^{1, 2, 3, 4, 5–6}. This study uncovers the molecular mechanism of substrate inhibition in tobacco glucosyltransferase NbUGT72AY1, which transfers glucose to phenols for plant protection. The peculiarity that β-carotene strongly attenuates the substrate inhibition of NbUGT72AY1, despite being a competitive inhibitor, allows to determine the conformational changes that occur during substrate binding in both active and substrate-inhibited complexes. Crystallography reveals structurally different ternary enzyme-substrate complexes that do not conform to classical mechanisms. An alternative pathway suggests substrates bind randomly, but the reaction occurs only if a specific order is followed (asymmetric cooperativity). This unreported paradigm explains substrate inhibition and reactivation by competitive inhibitors, opening new research avenues in metabolic regulation and industrial applications.