Abstract:
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.
摘要:
胰岛素降解酶(IDE)是一种高度保守的锌金属肽酶,能够催化裂解除胰岛素外的几种底物,在几种不同的生化途径中起着关键作用。虽然其作用机制已被广泛研究,许多难题仍然存在,阻碍了合理设计特异性调节剂的可能性,这些调节剂可能在糖尿病和阿尔茨海默病等几种疾病中具有重要的治疗应用。在这种情况下,我们开发了一种新颖的表面等离子体共振(SPR)方法,该方法允许在存在不同IDE活性调节剂的情况下直接测量胰岛素结合的酶协同性:肌肽,ATP,和EDTA。结果表明,IDE活性的正调制和负调制都可以与测得的Hill系数的增加和减少相关。分别,提供对IDE活动机制的新见解。寡聚化受阻的IDER767A突变体的使用证实,肌肽对IDE的正变构调节是由于固定在金SPR芯片上的酶也发生了酶寡聚状态的变化。