Quantenchemische Untersuchungen zur Hydrolyse von Formamid und zum Mechanismus von Carboxypeptidase A

In this work the mechanism of carboxypeptidase A is investigated using quantum chemical methods. In the context of enzymatic catalysis, the reactions of acyl derivates like esters and amides is of general importance. This work consists of two major parts. The first part concerns the hydrolysis of formamide. The reaction of formamide with water, OH- and H3O+ ions is investigated in the gas phase and in solution, including a continuum model. Additionally, the effect of two participating water molecules is taken into account. The reaction pathways for the hydrolysis of formamide have been used to carry out a benchmark study to allow for an evaluation of different density functional methods. The benchmark study results in averaged errors of up to 9 kcal/mol for the purely gradient corrected functionals and up to 5 kcal/mol for the hybrid functionals. Therefore, for the investigation of reaction mechanisms, the use of hybrid functionals is recommended. If no anionic species takes part in the reaction and computing time is an issue, a double zeta basis set including polarisation functions is sufficient for the calculation of relative energies. The parameterised CBS-QB3 method seems adequate to calculate exact thermodynamic data for organic reactions like the hydrolysis of formamide. The mechanisms of neutral, alkaline and acidic hydrolysis of formamide are addition-elimination-processes, proceeding via a tetrahedral intermediate. The reactions with hydroxide and hydronium ions are more favourable than the reaction with water. Taking into account the effect of assisting water molecules explicitly turns out to be essential. In the second part of this work the mechanism of catalytic hydrolysis of peptides by carboxypeptidase A is studied using a variation of model systems. The hydrolytic cleavage of formamid proceeds also via an addition-elimination-mechanism. Using the largest model system a reaction path with only one barrier could be found. The addition of the zinc-bound hydroxid is the only step requiring activation energy, thereby becoming rate-determining. Even if the relative energies of the transition states are underestimated by the density functional methods, the reaction path is energetically accessible. The consideration of explicit solvent effects is not necessary with the large model system, and the use of a continuum model has little influence on the relative energies of the calculated reaction pathways. A modified mechanism based on the proposal by Lipscomb is feasible. Glutamate-270 serves as a proton shuttle, and the carbonyl group of the amide/peptide bond is not only polarised by the zinc(II) ion, but also by the H-bridges from Arginine.