Computational and experimental exploration of low barrier hydrogen bonds and transition state models for serine esterase enzyme

The serine esterases acetylcholinesterase and butyrylcholinesterase are enzymes of importance to biomedicine, agriculture, and the military. The studies carried out herein examine the putative low-barrier hydrogen bonds that form in the active site during catalysis of the hydrolysis of substrate. Quantum mechanical calculations are used to construct a structural model for the transition state of the chemical step that forms a tetrahedral intermediate in the acylation stage of acetylcholinesterase-catalyzed hydrolysis of (acetylthio)choline. Finally, kinetic studies on butyrylcholinesterase catalyzed hydrolysis of (acetylthio)choline are conducted in attempts to characterize the transition state structure of this enzyme reaction as well.; Extensive quantum mechanical calculations of the structures and energetics of model low-barrier hydrogen bond systems locate the position of the hydrogen in the hydrogen bond. This was accomplished by computationally predicting the values of two independent physical observables, the nitrogen and proton chemical shifts, thereby locating the proton on the potential energy surface of the hydrogen bond. Experimental values for the Δδ 15N chemical shifts correlate how far the proton is transferred in each system.; Solvent and substrate deuterium kinetic isotope effect studies of butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine were conducted to gain insight into the catalytic mechanism and transition state structure of the enzyme. Experimentally, fractional rate limitations were determined and used along with free energy relationships to give the relative energetics of the sequential substrate binding and chemical steps in the acylation stage of catalysis.