Computer-based studies on enzyme catalysis : from structure to activity

L.O. Ridder

Research output: Thesisinternal PhD, WU


Theoretical simulations are becoming increasingly important for our understanding of how enzymes work. The aim of the research presented in this thesis is to contribute to this development by applying various computational methods to three enzymes of theβ-ketoadipate pathway, and to validate the models obtained by means of quantitative structure-activity relationships (QSAR). The models and the resulting QSARs provide valuable mechanistic information about the relevant (rate-limiting) steps in the reaction cycles of the enzymes studied.

Two of the enzymes that have been studied in this thesis, are flavin dependent monooxygenases: para -hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescence , and phenol hydroxylase (PH) from Trichosporon cutaneum . These enzymes catalyse the ortho-hydroxylation of para -hydroxybenzoate and phenol, leading to the formation of catechol and protocatechuate respectively. These products are the key intermediates in the degradation of many aromatic compounds. Once the catechol or protocatechuate is formed, the aromatic ring is cleaved between the two hydroxyl-substituted carbon atoms. This intradiol cleavage is catalysed by another enzyme studied in this thesis, catechol-1,2-dioxygenase (1,2-CTD), and by protocatechuate-3,4-dioxygenase (3,4-PCD), respectively.

Catechol dioxygenase

The reaction mechanism of catechol-1,2-dioxygenase from Pseudomonas putida has been studied by means of a QSAR approach based on gas-phase molecular orbital calculations. Catechol-1,2-dioxygenase catalyses intradiol cleavage of the aromatic ring of catechol by incorporating both oxygen atoms of molecular oxygen. In addition to the native catechol, this enzyme converts several C4-substituted catechol derivatives. In this study, the 4-methyl-, 4-fluoro-, 4-chloro-, 4-bromo, 4,5-difluoro- and 4-chloro,5-fluoro-catechols were obtained biosynthetically from the corresponding phenols by using the enzyme phenol hydroxylase. The overall rate constant for their conversion by catechol-1,2-dioxygenase was determined through steady-state kinetic experiments at various oxygen concentrations and saturating catechol concentrations.

The crucial step in the reaction mechanism of the enzyme catalysed reaction was considered to be the nucleophilic attack of the substrate on the oxygen molecule. Therefore, the experimental results were compared to calculated energies of the highest occupied molecular orbital (HOMO) of the various catechol substrates, which represent their nucleophilic reactivities. A (linear) correlation was found between the calculated HOMO energies and the logarithm of the experimental rate constants. This indicates that the rate-limiting step in the overall reaction cycle involves a nucleophilic reaction of the substrate. Thus, the reaction of the substrate with molecular oxygen may indeed be rate limiting. Additional calculations excluded two other steps in the reaction cycle as being rate limiting.

The results for catechol-1,2-dioxygenase from Pseudomonas putida were also compared to the data from two different types of catechol-1,2-dioxygenase, a normal (type I) and a chloro-catechol dioxygenase (type II), from Pseudomonas sp. B13. It could be argued that the difference in substrate preference between both types of catechol dioxygenases is related to a differential effect of the substituents on the rate of oxygen affinity binding by the two enzymes, rather than on the rate-limiting step.

p-Hydroxybenzoate hydroxylase

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.

The correlation also indicates that the QM/MM model provides a realistic description of the hydroxylation step, as it accounts correctly for the effect of substrate substituents on the rate of hydroxylation. This was the basis for a further and more detailed analysis of the QM/MM model, which provided detailed insight into the mechanism of substrate and cofactor activation to facilitate the electrophilic reaction. Deprotonation of the substrate, which has been observed experimentally, is shown to significantly lower the energy barrier for the calculated reaction pathway. Also, the QM/MM model allowed the analysis of the energetic effect of the individual amino acid residues on the hydroxylation reaction. The results suggest catalytic effects of a backbone carbonyl moiety (Pro293), by a specific stabilisation of the transition state, and of a (crystal) water molecule (Wat717), which stabilises the negative charge arising on the proximal oxygen of the flavin cofactor.

Phenol hydroxylase

The QM/MM technique has also been applied to phenol hydroxylase (PH). As for PHBH, the hydroxylation step, proposed to be rate limiting in the reaction cycle of PH, has been simulated for a series of halogenated substrate derivatives. The energy barriers obtained correlate well with the logarithm of the overall rate constants. This correlation supports that the electrophilic attack of the C4a-hydroperoxyflavin on the substrate is the rate-limiting step in the reaction cycle at pH 7.6 and 25°C. An additional mechanistic question addressed in this study is the protonation state of the substrate during hydroxylation. Substrate deprotonation as a mechanism of activation has not been established for PH as firmly as it has been for PHBH. Proton transfer from phenol to a potential active site base, Asp54, has been investigated by calculating a 2-dimensional potential energy surface for the two reaction coordinates, i.e. hydroxylation and proton transfer. This potential energy surface suggests that proton transfer prior to hydroxylation is the most favourable mechanism, which indicates that in the PH reaction substrate deprotonation is important as well. The QM/MM model was further analysed to provide insight into the effect of the protein environment on the simulated reaction steps. Some catalytic effects on the hydroxylation step, i.e. of a proline carbonyl moiety and of a crystal water in the active site of PH, were similar to those found for PHBH.

All together, the research presented in this thesis has made a new contribution to the development and validation of computational models that can be used to address a major challenge in the present field of biochemistry, i.e. to obtain insight into enzymatic reaction mechanisms and enzyme activity on the basis of the structure of enzyme and substrate(s). Special emphasis has been on the application and validation of the QM/MM technique in the context of a QSAR approach. The investigations of this thesis provide a first survey of the possibilities of the QM/MM method with respect to the prediction of biochemical activity, taking explicitly into account the influence of the active site surroundings.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Rietjens, Ivonne, Promotor
  • Vervoort, J., Promotor, External person
Award date3 Oct 2000
Place of PublicationS.l.
Print ISBNs9789058082527
Publication statusPublished - 2000


  • enzymes
  • catalysts
  • structure activity relationships
  • chemistry
  • quantum theory


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