β-Carotene alleviates substrate inhibition caused by asymmetric cooperativity

Enzymes play a crucial role in biological systems and biotechnological processes as vital and sustainable catalysts that facilitate and regulate biochemical reactions. Inhibition of product formation of an enzyme by its own substrate was originally dismissed as an artifact of biochemical analysis, but is now a proven fact and occurs in about 20% of known enzymes1. Substrate inhibition is attributed to the formation of an unproductive enzyme-substrate complex with no structural evidence of unproductivity provided to date1–6. Here we show in detail the molecular mechanism of substrate inhibition of the tobacco glucosyltransferase NbUGT72AY1, which transfers glucose from its nucleotide donor substrate to phenol acceptors as a plant protection measure. The peculiarity that the effector β-carotene strongly attenuates the substrate inhibition of NbUGT72AY1, although it unexpectedly acts as a competitive acceptor-substrate inhibitor, allowed us to uncover the conformational changes that occur during substrate binding in the active and substrate-inhibited complexes of this protein. X-ray crystallography revealed structurally different ternary enzyme-substrate complexes that do not conform to the classical compulsory ordered or random mechanism of an enzyme-catalyzed sequential bi-substrate reaction. We suggest an alternative pathway in which the two substrates bind randomly to the enzyme, but the reaction is only catalyzed if a specific order of substrate binding is observed (asymmetric cooperativity). The new paradigm explains substrate inhibition in monomeric multi-substrate enzymes and reactivation of activity by competitive inhibitors. This alternative concept opens up new avenues of research in metabolic regulation as well as a wealth of novel industrial applications. The results enable novel approaches, e.g. to minimize the effects of food components on drug metabolism and to study plant defense mechanisms from a new perspective and offer alternative approaches for the design of more efficient production processes.

酶作为不可或缺且可持续的催化剂，在生物系统与生物技术流程中发挥关键作用，可促进并调控生化反应。酶的自身底物对其产物生成的抑制作用最初被视作生化分析的人为假象，但如今已被证实为真实存在的现象，约20%的已知酶都会发生该现象¹。底物抑制被认为是由于无活性酶-底物复合物的形成所致，但截至目前尚无关于该复合物无活性的结构证据¹–⁶。本研究详细阐明了烟草糖基转移酶（glucosyltransferase）NbUGT72AY1的底物抑制分子机制：该酶可将核苷酸供体底物上的葡萄糖转移至酚类受体，这一过程是植物的一种防御机制。效应分子β-胡萝卜素可显著减弱NbUGT72AY1的底物抑制作用，尽管其意外地作为竞争性受体底物抑制剂发挥功能——这一特性使我们得以揭示该蛋白在活性状态与底物抑制状态复合物中，底物结合阶段发生的构象变化。X射线晶体学（X-ray crystallography）研究发现，存在结构各异的三元酶-底物复合物，此类复合物不符合酶催化顺序双底物反应的经典强制有序或随机结合机制。我们提出了一种全新的反应路径：两种底物可随机结合至酶，但仅当底物结合遵循特定顺序时，反应才会被催化（不对称协同性）。这一新范式可解释单体多底物酶中的底物抑制现象，以及竞争性抑制剂对酶活性的复活作用。这一全新概念为代谢调控领域的研究开辟了新方向，同时也为众多新兴工业应用提供了思路。本研究成果为减少食品成分对药物代谢的影响提供了全新思路，也为从新视角研究植物防御机制提供了可能，并为设计更高效的生产流程提供了替代性方案。