Catalysis of amide isomerization: Investigations into the possible mechanisms of action of peptidyl prolyl isomerase enzymes

Peptidyl prolyl isomerases (PPIases), “rotamase” enzymes that catalyze the cis-trans isomerization of amides, are important clinically as the biological receptors for the immunosuppressive drugs cyclosporin A and FK-506, and have been proposed to play many roles in the body, including: catalysis of protein folding; functioning as auxiliary enzymes in HIV-1 protease-mediated reactions; modulation of calcium release; and mitotic regulation. Although these enzymes have been well studied, a detailed understanding of the tactics they utilize to catalyze amide isomerization remains elusive, as do many of their biological functions. Full knowledge of how the PPIases work at the molecular level is desirable for the development of inhibitors of the enzymes to allow further mapping of their roles in vivo, as well as to aid in the design of fully synthetic catalysts of protein folding in vitro. This dissertation describes the first experimental verification (in model systems) of several mechanisms proposed to catalyze amide isomerization in the PPIase active site, as well as preliminary work toward the goal of developing in vitro catalysts of protein folding.;Following a detailed introduction to the amide functionality and the PPIase enzymes, Chapter 2 describes a novel intramolecular hydrogen bond that is proposed to play an important role in the enzymatic catalysis of amide isomerization (AI). Chapters 3 and 4 extend this concept to the first system in which the amide nitrogen acts as an acceptor atom for a moderately strong hydrogen bond, and shows that this interaction efficiently catalyzes AI. In Chapter 5, we present the first example of nucleophilic catalysis of AI, while Chapter 6 describes the first well documented examples of metal-catalyzed AI, along with novel spectroscopic and crystallographic data that suggest metal coordination to the amide nitrogen as the catalytically relevant interaction. Also, we show that Cu(II) ions can catalyze the folding of a small model protein in vitro. Finally, Chapter 7 describes a study of solvent effects on the barrier to rotation in carbamates, and presents a comparison to the analogous process in amides.