How and When Does an Enzyme React? Unraveling α-Amylase Catalytic Activity with Enhanced Sampling Techniques

Enzymatic catalysis is a complex process that can involvemultipleconformations of the enzyme:substrate complex and several competitivereaction pathways, resulting in a multi-dimensional free energy landscape.The study of enzymatic activity often requires deep knowledge of thesystem to establish the catalytic mechanism and identify the possiblereactive conformations of the complex. Here, we present an enhancedsampling and machine learning-based approach to explore the catalyticreaction space and characterize the transformation from reactive tonon-reactive conformations with minimal a priori knowledgeof the system. We applied this approach to study the rate-determiningstep of the glycolysis reaction of maltopentose catalyzed by humanpancreatic alpha-amylase, an important enzyme in glucose productionas well as a major drug target for the treatment of type-II diabetes.We unravel the complexity of the enzymatic reaction, reveal threebinding modes of the substrate within the active site, and highlightthe role of water in the catalytic process and in the stepwise conversionof reaction-ready to non-reactive conformations. Overall, these insightsoffer atomistic details on the catalytic mechanism and dynamics ofthe active site, allowing one to shed light on two fundamental questionsin enzymatic catalysis, that is how and when does an enzyme react?

Automated summary

In this study, an enhanced sampling and machine learning-based approach was used to explore the catalytic reaction space and characterize the transformation from reactive to non-reactive conformations. By studying the rate-determining step of the glycolysis reaction, we unraveled the complexity of enzymatic reaction, revealed three binding modes of the substrate, and highlighted the role of water in the catalytic process and in the stepwise conversion of reaction-ready to non-reactive conformations.