Chemical Science Department, University of Catania, Viale Andrea Doria 6, Catania, 95125, Italy.
Anal Bioanal Chem. 2022 Jul;414(17):4793-4802. doi: 10.1007/s00216-022-04122-3. Epub 2022 May 16.
Insulin-degrading enzyme (IDE) is a highly conserved zinc metallopeptidase and is capable to catalytically cleave several substrates besides insulin, playing a pivotal role in several different biochemical pathways. Although its mechanism of action has been widely investigated, many conundrums still remain, hindering the possibility to rationally design specific modulators which could have important therapeutical applications in several diseases such as diabetes and Alzheimer's disease. In this scenario, we have developed a novel surface plasmon resonance (SPR) method which allows for directly measuring the enzyme cooperativity for the binding of insulin in the presence of different IDE activity modulators: carnosine, ATP, and EDTA. Results indicate that both positive and negative modulations of the IDE activity can be correlated to an increase and a decrease of the measured Hill coefficient, respectively, giving a new insight into the IDE activity mechanism. The use of the IDE R767A mutant for which oligomerization is hindered confirmed that the positive allosteric modulation of IDE by carnosine is due to a change in the enzyme oligomeric state occurring also for the enzyme immobilized on the gold SPR chip.
胰岛素降解酶（IDE）是一种高度保守的锌金属肽酶，除了胰岛素之外，还能够催化切割几种底物，在几种不同的生化途径中发挥关键作用。尽管其作用机制已被广泛研究，但仍存在许多未解之谜，这阻碍了合理设计特定调节剂的可能性，而这些调节剂在糖尿病和阿尔茨海默病等多种疾病的治疗中可能具有重要的应用价值。在这种情况下，我们开发了一种新的表面等离子体共振（SPR）方法，该方法可直接测量在不同 IDE 活性调节剂（肌肽、ATP 和 EDTA）存在下胰岛素结合的酶协同性：结果表明，IDE 活性的正调控和负调控分别与测量的Hill 系数的增加和减少相关，这为 IDE 活性机制提供了新的见解。使用酶的 R767A 突变体，该突变体阻碍了寡聚化，证实了肌肽对 IDE 的正变构调节是由于酶的寡聚状态发生变化，这种变化也发生在固定在金 SPR 芯片上的酶上。
