Modeling the Complete Catalytic Cycle of Aspartoacylase

Abstract

The complete catalytic cycle of aspartoacylase (ASPA), a zinc-dependent enzyme responsible for cleavage of N-acetyl-l-aspartate, is characterized by the methods of molecular modeling. The reaction energy profile connecting the enzyme-substrate (ES) and the enzyme-product (EP) complexes is constructed by the quantum mechanics/molecular mechanics (QM/MM) method assisted by the molecular dynamics (MD) simulations with the QM/MM potentials. Starting from the crystal structure of ASPA complexed with the intermediate analogue, the minimum-energy geometry configurations and the corresponding transition states are located. The stages of substrate binding to the enzyme active site and release of the products are modeled by MD calculations with the replica-exchange umbrella sampling technique. It is shown that the first reaction steps, nucleophilic attack of a zinc-bound nucleophilic water molecule at the carbonyl carbon and the amide bond cleavage, are consistent with the glutamate-assisted mechanism hypothesized for the zinc-dependent hydrolases. The stages of formation of the products, acetate and l-aspartate, and regeneration of the enzyme are characterized for the first time. The constructed free energy diagram from the reactants to the products suggests that the enzyme regeneration, but not the nucleophilic attack of the catalytic water molecule, corresponds to the rate-determining stage of the full catalytic cycle of ASPA.

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

“…Both monomers, ASPA and ASPB, contain the active site and the gates. Figure 1 was drawn by motifs of the crystal structure PDB ID: 2O53 6 and the results of the quantum mechanics/molecular mechanics (QM/MM) modeling 11 consistent with the experimental findings. It is important to note that the gates to the active sites are closed in the crystal structure of the apo-protein in the sense that short hydrogen-bond distances between the pairs Arg71-Glu293 and Tyr64-Lys291 are observed.…”

Demystifying DPP III Catalyzed Peptide Hydrolysis—Computational Study of the Complete Catalytic Cycle of Human DPP III Catalyzed Tynorphin Hydrolysis

“…In the final EP structure, the C-product monodentately coordinates the Zn 2+ (with O w atom), and the N-product, in its neutral form, is hydrogen-bonded to the oxygen atom, coordinating the metal ion (O w -H w2 distance 2.23 Å) and to the protonated E451 (N s -H w1 distance 1.58 Å). In other hydrolytic enzymes such as aspartoacylase, a very flat energy surface has been observed in the final phase of the reaction due to the fluctuation of the system between a few conformations [29]. These conformations differ in whether the C-product forms a hydrogen bond with the protonated amine group of the N-product and the catalytic glutamate is deprotonated, or whether the C-product remains separated from the N-product and the catalytic glutamate is protonated.…”

“…The experimental data suggest otherwise. It is known that, in addition to chemical transformation, Michaelis complex formation and/or product release can be the ratelimiting steps in the complete catalytic cycle of an enzyme, as shown for metalloproteinase-2 and aspartoacylase [28,29]. To gain a more complete insight into DPP III substrate specificity, it is very important to understand the overall catalytic cycle of the enzyme.…”

Kinetics of chemical processes in the human brain. Trigger effect and self-stabilization of N-acetylaspartic acid

“…Молекулярно-кинетическое исследование этих эффектов на основе QM/MM приближений дано нами в работах [9][10][11][12].…”

“…1, являются решением системы уравнений (1)-(5) при следующих значениях параметров: α = 6; β = 1; A = 0,135 • 10 -5 ; ξ = 1; n = 6; S 0 = 0,008; k 1 = 11,5; k 2 = 0,14; k 3 = 102; a 0 = 0,248; b 0 = 1,8 • 10 -13 ; b 1 = 9 • 10 -9 ; b 2 = 4,5 • 10 -4 ; b 3 = 2; b 4 = 2,44 • 10 3 . Значения a 0 , b 0 , b 1 , b 2 , b 3 и b 4 определены из экспериментальных данных в соответствии с [8][9][10][11][12]. Важно отметить, что максимальное значение концентрация кислорода достигает на 6-й секунде после внешнего раздражителя, минимальное значение N-ацетиласпартата -на 12-й секунде.…”