Computer-based Studies on Enzyme Catalysis: From Structure to Activity

An important step that has been made in this thesis is the use of a combined quantum mechanical/molecular mechanical (QM/MM) method. Using this method, the quantum mechanical (reaction pathway) calculation of the reacting compounds could be performed within the actual environment of the protein. The surrounding protein atoms are calculated at a molecular mechanical (MM) level and their electrostatic and steric effects on the quantum mechanical system are included. This QM/MM technique has been applied to the hydroxylation step catalysed by p -hydroxybenzoate hydroxylase (PHBH). It was first investigated whether the energy barriers obtained from QM/MM reaction pathway calculations could be used to explain the variation in the overall rate constants for the conversion of a series of fluorinated substrates by PHBH. Reaction pathways were calculated for the proposed rate-limiting step in the reaction cycle: the electrophilic attack of the C4a-hydroxyperoxyflavin cofactor intermediate on the substrate. The energy profiles calculated for this reaction step with the various substrates yielded barriers with different heights. A correlation was found between the natural logarithm of the experimental overall rate constants for conversion of the fluorinated substrates by PHBH and the QM/MM calculated energy barriers for the different substrates. This correlation with overall rate constants supports that the electrophilic attack of the C4a-hydroxyperoxyflavin on the substrate is indeed the rate-limiting step in the reaction cycle.