Date of Award

Fall 2011

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biomedical Engineering

First Advisor

Daniela S. Mainardi


Recent advancement in enzyme catalysis has opened ways to design efficient biocatalysts, bio-sensors and bio-fuel cells. An in-depth knowledge about the mechanism of the reaction taking place within the enzymes is of great importance to achieve these goals. In this dissertation, various computation methods are applied to investigate the mechanism behind enzyme catalysis in the presence of compounds called activators.

Methanol dehydrogenase (MDH) is a well-known bio-catalyst that can oxidize excess of methanol from the environment to formaldehyde. The enzyme works well within the bacterial environment, but under in vitro, it loses activity. Ammonia is used as an activator to restore the activity of MDH. The Monte Carlo search using simulated annealing metaheuristic method is conducted to explore the binding of MDH with its natural electron acceptor Cytochrome cL in varying concentration of ammonia. The main aim behind this is to explore the interaction energy between the enzymes under the influence of its activator. The concentration of ammonia is varied from 0 to 5 ammonia molecules.

Moving deeper into the active site of MDH, molecular mechanics and dynamics calculations were performed to investigate the position and effect of ammonia in the active site amino acids of MDH. The concentration of ammonia was varied from 0 to 55.39 mM. It was proposed that ammonia may form a complex conjugate with the cofactor of MDH (Pyrroloquinoline quinone) to assist in the oxidation of methanol. Two of the most debated methanol oxidation mechanisms, Addition-Elimination reaction and Hydride-Transfer mechanism, were used to investigate the role of ammonia in the oxidation of methanol. Density functional theory (DFT) was applied to explore the methanol oxidation mechanism in the presence of ammonia. Models of varying size that best represent the active site of MDH were tested for this purpose.

The interaction energy obtained after the docking of MDH and Cytochrome cL (CL) indicate that the presence of a single ammonia molecule increased the binding between the enzymes by 108.50 kcal/mol. The presence of ammonia at the active site of MDH affected the diffusivity of various components of the active site, such as the Ca2+ ion, Glu177, Asn261 and probable catalytic base Asp303. Rise in the concentration of ammonia increased the diffusivity coefficient of active site components. At ammonia concentration of 20 mM to 35 mM, the distance among Pyrroloquinoline quinine (PQQ), Ca2+ and Asp303 decreased, which assisted in the oxidation of methanol. Excess of ammonia caused instability in the amino acids of the active site and increased the distance between the cofactor and Asp303, thereby reducing the affinity of MDH towards methanol.

Based on the transition states obtained, it is observed that ammonia does not form any complex with PQQ of MDH during the oxidation mechanism. Instead, ammonia assisted in the re-oxidation of PQQ by abstracting a proton from the reduced form of PQQ (PQQH2). Re-oxidation of PQQ was important as it allowed the oxidation of subsequent methanol molecule, thus increasing the activity and efficiency of the enzyme. The free energy barriers of the rate determining step indicated that the ammonia reduced the activation energy involved in the dissociation of the methanol molecule by 6.2 kcal/mol in the case of Addition-Elimination reaction and 6.8 kcal/mol in the case of Hydride-Transfer mechanism.