Mechanistic Insights into Dinuclear Zinc Enzymes from Density Functional Theory Studies

In this thesis, quantum chemical methods have been used to shed light on the reaction mechanisms of several dinuclear zinc enzymes. The enzymes studied are involved in the hydrolysis of phosphates, amides, and carboxylic esters, namely RNase Z, Dihydroorotase (DHO), and N-acyl homoserine lactone hydrolase (AHL lactonase). The density functional method B3LYP, together with quite large active site models, was used to investigate these enzymatic reactions. Several plausible proposed mechanisms, involving protonation states of important active site residues (DHO), substrate orientations (AHL lactonase), have been considered. The calculated energetics can be used to assess the feasibility of the suggested reaction mechanisms. Based on the calculations and also on other related dinuclear zinc enzymes studied previously, some general mechanistic features have been uncovered. For all three enzymes, the nucleophilicity of the bridging hydroxide is shown to be sufficient to perform the nucleophilic attack on the substrates. During the attack, the negative charge is transferred from the bridging hydroxide to the substrate oxygen (P=O or C=O). For phosphate hydrolysis, an in line attack have been suggested for RNase Z. In addition, the two zinc ions in RNase Z are directly involved in stabilizing the negative charge in the penta-coordinated transition states. For carbonyl substrates, only one zinc ion participates in the oxygen anion stabilization in the transition states and the tetrahedral intermediates. Furthermore, the enzymes always use the zinc ion with less negatively-charged ligands to play such role. All the substrates investigated have poor leaving groups. Therefore, either the zinc ions or some active site residues help the cleavage of the scissile bond. For RNase Z, a Glu-His diad was suggested to protonate the leaving group. For DHO, an Asp residue was shown to transfer a proton from the bridging hydroxide to the leaving group nitrogen. For AHL lactonase, a zinc ion was also observed to stabilize the leaving oxygen anion.