The Effect of Mutations on Fast Protein Dynamics

Abstract

Enzymes are powerful natural catalysts that can greatly enhance the rate of chemical reactions. This rate acceleration is crucial to all life forms on earth. Understanding how enzymes work has been one of the major focus in biochemistry. In this dissertation, I present three studies targeted specifically at fast sub-picosecond protein motions that are directly coupled to the reaction coordinate of the chemical step in the enzyme catalytic process, which are called the rate promoting vibration. This type of motion has been found in many enzyme systems in previous studies. The three studies presented in this dissertation focused on three enzymes and their variants: the directed evolution process of KE59, Catechol O-Methyltransferase and its Y68A mutant, and the Morphinone Reductase and its N189A mutant. Detailed knowledge of the behavior of the rate promoting vibration in the three enzyme systems and their variants has been fully discussed in this dissertation. In addition, through comparisons between enzymes that possess rate promoting vibrations and their mutagenesis variant that do not, I found that active site mutations, even though not directly involved in any protein motions, can significantly affect existing rate promoting vibrations. The rate promoting vibration is so sensitive to the active site geometry that subtle influences on the active site geometry induced by even a single point mutation can eliminate its effect, hence making it decoupled from the chemical step of the enzymatic reaction. Also, we have shown in the second project that the rate promoting vibration creates a more reaction-favored active site electric field, reconciling the ongoing debate between the fast protein motions proposal and the electrostatic pre-organization proposal in explaining the origin of enzyme catalytic power. This knowledge is of great value to the on-going effort in altering existing rate promoting vibrations, or building rate promoting vibrations into enzymes to enhance their catalytic efficiency.