Computational Analysis of the Inhibition Mechanism of NOTUM by the ONIOM Method.

Notum is a member of serine hydrolyses that cleaves the palmitoleate moiety from Wingless-related integration site (Wnt) ligands. This enzyme plays crucial functions through modulating the Wnt signaling pathway. Inhibition of Notum carries therapeutic effects against a number of maladies including osteoporosis, cancer, and Alzheimer's disease. Recently, a class of irreversible inhibitors based on esters of 4-(indolin-1-yl)-4-oxobutanoic acid have been reported. Using the crystal structures of enzyme-4-(indolin-1-yl)-4-oxobutanoate adduct and 4-(indolin-1-yl)-4-oxobutanoic acid-enzyme complex, we studied computationally the proposed inhibition mechanism using model systems based on the own n-layered integrated molecular orbital and molecular mechanics (ONIOM) method. In the first place, model systems were formulated to investigate the transesterification between the catalytic serine residue, Ser-232, and the methyl ester of 4-(indolin-1-yl)-4-oxobutanoate. In the second place, the hydrolysis mechanism of the resultant enzyme-inhibitor adduct was studied. The energetics of these steps were analyzed using a density functional theory functional in the ONIOM method. In addition, the roles of active-site residues during these steps were highlighted. It was found that the hydrolysis of the covalent adduct is highly endergonic corroborating the irreversible inhibition mechanism. These results will shed light not only on the inhibition mechanism but also on the catalytic mechanism.

Computational Analysis of Histone Deacetylase 10 Mechanism by the ONIOM Method: A Complementary Approach to X-ray and Kinetics Studies.

Histone deacetylase 10 (HDAC 10) catalyzes deacetylation of N8-acetylspermidine into spermidine in the cytosolic region of eukaryotic cells. Inhibition of HDAC 10 has clinical importance in certain types of cancers. Recently, X-ray crystal structures corresponding to the substrate-bound, tetrahedral intermediate-bound, and product-bound enzymes have been resolved using variant forms of humanized HDAC 10. Based on these structures, it was proposed that Y307 residue polarizes the carbonyl of the acetyl group in N8-acetylspermidine together with a zinc atom, which is coordinated by D174, H176, D267, and an H2O molecule. The H2O molecule undergoes nucleophilic addition to the carbonyl carbon of N8-acetylspermidine to form the tetrahedral intermediate. During this process, it is suggested that H136 acts as a general base to deprotonate the H2O molecule. It is further proposed that the protonation of the amide N atom of the tetrahedral intermediate by H137 causes the deacetylation forming the final products, spermidine and acetate ion. In this study, computational models based on the ONIOM method were employed to study the proposed mechanism for the two steps of the deacetylation process based on the crystal structure of the substrate-bound enzyme. The energy profiles of each step as well as the roles of the active site residues were investigated for the catalysis. The calculated activation barrier is in good agreement with the reported kcat value.

Mechanistic study of L-6-hydroxynicotine oxidase by DFT and ONIOM methods.

L-6-Hydroxynicotine oxidase (LHNO) is a member of monoamine oxidase (MAO) family and catalyzes conversion of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during bacterial degradation of nicotine. Recent studies indicated that the enzyme catalyzes oxidation of carbon-nitrogen bond instead of previously proposed carbon-carbon bond. Based on kinetics and mutagenesis studies, Asn166, Tyr311, and Lys287 as well as an active site water molecule have roles in the catalysis of the enzyme. A number of studies including experimental and computational methods support hydride transfer mechanism in MAO family as a common mechanism in which a hydride ion transfer from amine substrate to flavin cofactor is the rate-limiting step. In this study, we formulated computational models to study the hydride transfer mechanism using crystal structure of enzyme-substrate complex. The calculations involved ONIOM and DFT methods, and we evaluated the geometry and energetics of the hydride transfer process while probing the roles of active site residues. Based on the calculations involving hydride, radical, and polar mechanisms, it was concluded that hydride transfer mechanism is the only viable mechanism for LHNO.