Quantum Chemical Studies of Enzymatic Reaction Mechanisms : Investigations of Cytosine Deaminase and ω-Transaminase

Abstract: In this thesis, density functional theory is used to study the reaction mechanisms of two dierent enzymes. Quantum chemical cluster models of the active sites were designed using available crystal structures. In this approach only the active site residues are considered and the effects of the surroundings are accounted for by a coordinate-locking scheme and a polarizable continuum model.The enzymes studied are cytosine deaminase (CDA) from Escherichia coli and ω-transaminase from Chromobacterium violaceum (Cv-ωTA). CDA is a zinc-dependentenzyme that catalyzes the hydrolytic deamination of cytosine into uracil and ammonia. Cv-ωTA carries out the interchange of amino and keto groups by utilizing the cofactor pyridoxal-5’-phosphate (PLP). The calculations provide optimized geometries and energies of transition states and intermediates, which are analyzed and used to construct a potential energy prole for the reaction and to identify the rate-limiting step. Each theoretical investigation provides a detailed description of the catalytic mechanism and establishes the roleof important active site residues.In the rst study (Paper I), it was found that a glutamate and an aspartate residue assist in the proton transfer events throughout the reaction. In the second study (Paper II), it was found that the lysine residue, which in the holo enzyme binds the cofactor PLP, assists in several proton transfer events once it has been replaced by the amino substrate. It was also found that the water substrate can be utilized as a proton shuttle before it is consumed at a later stage in the reaction mechanism.Apart from the detailed chemical insight, the results in this thesis confirmthat density functional theory together with cluster models of active sites is a very useful approach for studying diverse enzymatic reaction mechanisms.