Assessing the Catalytic Role of Native Glucagon Amyloid Fibrils

Glucagon stands out as a pivotal peptide hormone, instrumental in controlling blood glucose levels and lipid metabolism. While the formation of glucagon amyloid fibrils has been documented, their biological functions remain enigmatic. Recently, we demonstrated experimentally that glucagon amyloid fibrils can act as catalysts in several biological reactions, including esterolysis, lipid hydrolysis, and dephosphorylation. Herein we present a multiscale quantum mechanics/molecular mechanics (QM/MM) simulation of the acylation step in the esterolysis of para-nitrophenyl acetate (p-NPA), catalyzed by native glucagon amyloid fibrils, serving as a model system to elucidate their catalytic function. This step entails a concerted mechanism, involving proton transfer from serine to histidine, followed by the nucleophilic attack of serine oxy anion on the carbonyl carbon of p-NPA. We computed the binding energy and free-energy profiles of this reaction using the PDLD/S-LRA-2000 and the empirical valence bond (EVB) methods. This included simulations if the reaction in an aqueous environment and in the fibril, enabling us to estimate the catalytic effect of the fibril. Our calculations obtained a barrier of 23.4 kcal∙mol–1 for the enzyme-catalyzed reaction, compared to the experimental value of 21.9 kcal∙mol–1 (and a calculated catalytic effect of 3.2 kcal∙mol–1 compared to the observed effect of 4.7 kcal∙mol–1) This close agreement together with the barrier reduction when transitioning from the reference solution reaction to the amyloid fibrils provides supporting evidence to the catalytic role of glucagon amyloid fibrils. Moreover, by employing the PDLD/S-LRA-2000 approach further reinforced exclusively the enzyme's catalytic role. The results presented in this study contribute significantly to our understanding of the catalytic role of glucagon amyloid fibrils, marking, to the best of our knowledge, the first mechanistic investigation of fibrils using QM/MM methods. Therefore, our findings offer fruitful insights for future research on the mechanisms of related amyloid catalysis.