Mechanistic Pathway on Human α‑Glucosidase Maltase-Glucoamylase Unveiled by QM/MM Calculations

The
excessive consumption of starch in human diets is associated
with highly prevalent chronic metabolic diseases such as type 2 diabetes
and obesity. α-Glucosidase enzymes contribute to the digestion
of starch into glucose and are thus attractive therapeutic targets
for diabetes. Given that the active sites of the various families
of α-glucosidases have different sizes and structural features,
atomistic descriptions of the catalytic mechanisms of these enzymes
can support the development of potent and selective new inhibitors.
Maltase-glucoamylase (MGAM), in particular, has a N-terminal catalytic
domain (NtMGAM) that has shown high inhibitor selectivity. We provide
here the first theoretical study of the human NtMGAM catalytic domain,
employing a hybrid QM/MM approach with the ONIOM method to disclose
the full atomistic details of the reactions promoted by this domain.
We observed that the catalytic activity follows the classical Koshland
double-displacement mechanistic pathway that uses general acid and
base catalysts. A covalent glycosyl-enzyme intermediate was formed
and hydrolyzed in the first and second mechanistic steps, respectively,
through oxocarbenium ion-like transition state structures. The overall
reaction is of dissociative type. Both transition state geometries
differ from those known to occur in other glycosidases. The activation
free energy for the glycosylation rate-limiting step agrees with the
experimental barrier of 15.8 kcal·mol–1. Both
individual mechanistic steps of the reaction are exoergonic. These
structural results may serve as the basis for the design of transition
state analogue inhibitors that specifically target the intestinal
NtMGAM catalytic domain, thus delaying the production of glucose in
diabetic and obese patients.

人体饮食中过量摄入淀粉，与2型糖尿病、肥胖症等高发慢性代谢疾病密切相关。α-葡糖苷酶（α-Glucosidase）可将淀粉降解为葡萄糖，因此成为糖尿病治疗的热门靶点。鉴于不同家族的α-葡糖苷酶其活性位点具有各异的尺寸与结构特征，对这类酶催化机制的原子级描述，可为开发强效且具备选择性的新型抑制剂提供重要支撑。麦芽酶-葡糖淀粉酶（Maltase-glucoamylase，MGAM）的N端催化结构域（NtMGAM）已被证实具有较高的抑制剂选择性。本研究首次针对人类NtMGAM催化结构域开展理论研究，采用结合ONIOM方法的混合量子力学/分子力学（QM/MM）途径，揭示该结构域所催化反应的完整原子级细节。研究发现，其催化活性遵循经典的Koshland双置换催化机理，该机理依赖广义酸碱催化剂。反应的两个独立机理步骤分别通过类氧碳鎓离子（oxocarbenium ion）过渡态结构，依次完成共价糖苷-酶中间体的形成与水解。整体反应属于解离型反应，且两种过渡态的几何结构均不同于其他糖苷酶（glycosidases）中已知的过渡态结构。糖基化限速步骤的活化自由能与15.8 kcal·mol–1的实验能垒相符。该反应的两个单独机理步骤均为放能反应。上述结构研究结果，可为设计靶向肠道NtMGAM催化结构域的过渡态类似物抑制剂提供理论依据，从而延缓糖尿病与肥胖症患者体内葡萄糖的生成。