The catalytic mechanism of phospholipase C and the total synthesis of erythromycin B

Phospholipase C (PLC) is an important enzyme in the signal transduction cascade, and as such its mechanism of action has long been the subject of scrutiny. However, until recently little progress had been made in discerning the mode of action of PLC. Herein is described research elucidating the catalytic mechanism of phospholipase C (Bacillus cereus) utilizing the combined tools of biochemistry and molecular biology. Through these studies, the roles of many active site amino acid residues have been determined. In addition, the catalytic cycle of PLC has been conclusively established, including the rate-determining step and order of product release.;In the course of these investigations, several new experimental techniques and protocols needed to be developed. Thus, two new assays for determining the kinetic parameters of PLC are described. These assays were invaluable throughout the course of this research, and have since been applied to another phospholipase as well. In addition, new vectors for recombinant protein expression and a novel means to probe zinc enzymes are detailed.;Phospholipase C is known to preferentially hydrolyze phosphatidylcholine (PC) substrates, although it also processes phosphatidylethanolamine (PE) and phosphatidylserine (PS) at reduced rates. One goal of this research was to engineer PLC such that it now processed PE or PS faster than PC. This was accomplished by randomly mutating three amino acid residues and subsequently screening E. coli colonies that harbor these mutant proteins. In this manner, both PE and PS selective enzymes were created.;The erythromycin family of macrolide antibiotics have long been of interest to synthetic chemist due to there clinical efficacy and unique molecular architecture. The final chapter in this dissertation details the total synthesis of erythromycin B. This was achieved via a novel strategy, one that saves synthetic steps and could be a viable alternative to traditional methods for macrolide synthesis.