Three Faces of N-Acetylaspartate: Activator, Substrate, and Inhibitor of Human Aspartoacylase

Abstract

Hydrolysis of N-acetylaspartate (NAA), one of the most concentrated metabolites in brain, catalyzed by human aspartoacylase (hAsp) shows a remarkable dependence of the reaction rate on substrate concentration. At low NAA concentrations, sigmoidal shape of kinetic curve is observed, followed by typical rate growth of the enzyme-catalyzed reaction, whereas at high NAA concentrations self-inhibition takes place. We show that this rate dependence is consistent with a molecular model, in which N-acetylaspartate appears to have three faces in the enzyme reaction, acting as activator at low concentrations, substrate at moderate concentrations, and inhibitor at high concentrations. To support this conclusion we identify binding sites of NAA at the hAsp dimer including those on the protein surface (activating sites) and at the dimer interface (inhibiting site). Using the Markov state model approach we demonstrate that population of either activating or inhibiting site shifts the equilibrium between the hAsp dimer conformations with the open and closed gates leading to the enzyme active site buried inside the protein. These conclusions are in accord with the calculated values of binding constants of NAA at the hAsp dimer, indicating that the activating site with a higher affinity to NAA should be occupied first, whereas the inhibiting site with a lower affinity to NAA should be occupied later. Application of the dynamical network analysis shows that communication pathways between the regulatory sites (activating or inhibiting) and the gates to the active site do not interfere. These considerations allow us to develop a kinetic mechanism and to derive the equation for the reaction rate covering the entire NAA concentration range. Perfect agreement between theoretical and experimental kinetic data provides strong support to the proposed catalytic model.

Dynamics of the Multipathway Regulation of the Vasodilator Bold Effect Induced by a Nerve Impulse: A Kinetic Model of the Neurovascular Coupling Process

“…Particularly characteristic manifestations of molecular polymorphisms of human proteins are related to the functioning of cholinesterase and acylase enzymes. − The structural diversity of the catalytic activity of these enzymes provides a variety of dynamic responses in the same processes. Molecular polymorphisms are the basis of the predisposition to certain diseases. ,− …”

Structure of the Brain N-Acetylaspartate Biosynthetic Enzyme NAT8L Revealed by Computer Modeling

“…Additional large-scale molecular dynamics and quantum-based calculations are required to clarify the reaction mechanism and the role of every residue in NAT8L. The strategy applied earlier − to model NAA hydrolysis by ASPA can be used in subsequent studies to understand details of the NAA kinetics in cells. Simulations described in this work allow us to reveal the principal features in the active site of the NAT8L enzyme, the structure of which is unknown from experimental studies.…”

Computational Structural Biology: Successes, Future Directions, and Challenges

“…Among the tenets that computational structural biology may increasingly embrace in the next few years are integrated deep neural networks, Markov state models, and metastable states for sampling conformational space, which will permit mining the entire free-energy landscape, thus providing insight into biological macromolecular actions, including catalysis [123,124,125,126,127,128,129] and dynamic networks [130]. Other topics of interest are integrative or hybrid modeling across disparate scales [131,132,133,134,135,136], including organelles, cells, and tissues, and archiving of the models [137]; harnessing machine intelligence to extract trends and predict outcomes; making headway in precision medicine [138,139]; improving software for imaging technologies and analysis and unveiling the mechanisms, on the structural level, through which the microbiota can hijack host signaling and impact human health.…”