The Dynamics of Dehydrogenases - A Phase Space Odyssey

Enzymes are immensely powerful and efficient heterogenous catalysts which are essential for life. As essential to life as enzymes are, it is still not well understood exactly how they enhance the rate of their catalyzed reactions up to 19 orders of magnitude over their solution phase counterpart reactions. Recent research has focused on sub--picosecond motions coupled to the reaction coordinate, called rate--promoting vibrations, which are important components of several well--known enzymatic mechanisms and build upon previous models of enzyme activity. Herein I present two studies which are expressly focused on providing tools and knowledge to understand how dynamics affects enzymatic reactions. First, I present a method for the calculation of kinetic isotope effects from first principles, using transition path sampling and centroid molecular dynamics. This method allows for the calculation of kinetic isotope effects without the assumptions necessitated by transition state theory or free energy perturbation methods. It was found that this method could calculate the primary H/D kinetic isotope effect of the conversion of benzyl alcohol to benzaldehyde in yeast alcohol dehydrogenase to within the margin of error of experimentally measured kinetic isotope effects of the same reaction. Second, I examined the role that evolution plays in the preservation of these rate--promoting vibrations, by performing a transition path sampling study of two lactate dehydrogenases, those of Plasmodium falciparum and Cryptosporidium parvum, which evolved through separate gene duplication events from a common malate dehydrogenase ancestor. It was found that though both lactate dehydrogenases share the same rate--promoting vibration, and indeed share the rate--promoting vibration found in other lactate dehydrogenases, the sequence variations in lactate dehydrogenase from P. falciparum causes a diminished contribution of the motions to the reaction coordinate. The studies presented in this dissertation contribute to the our understanding of enzymes on an atomistic level, as well as providing tools necessary for designing novel de novo enzymes and targeted drugs for enzymes of disease--causing organisms