Peptide hormones are responsible for cellular functions critical to the survival of an organism. Approximately 50% of all known peptide hormones are post-translationally modified at their C-terminus. Peptidylglycine alpha-amidating monooxygenase (PAM) is a bi-functional enzyme which catalyzes the activation of peptide pro-hormones. PAM also functionalizes long chain N-acylglycines suggesting a potential role in signaling as their respective fatty acid amides. As chain length increases for N-acylglycines so does the catalytic efficiency. This effect was probed further by primary kinetic isotope effects and molecular dynamics to better resolve the mechanism for improved catalytic function. The 1°KIE showed a linear decrease with increasing chain length. Neither the minimal kinetic mechanism nor the maximal rate for substrate oxidation was observed to be altered by substrate hydrophobicity. It was concluded that KIE suppression was a function of 'Pre-organization' - more efficient degenerate wave function overlap between C-H donor and Cu(II)-superoxo acceptor with increased chain length. Substrate activation is believed to be facilitated by a Cu(II)-superoxo complex formed at CuM. Benzaldehyde imino-oxy acetic acid undergoes non-enzymatic O-dealkylation to the corresponding oxime and glyoxylate products. This phenomena was further studied using QM/MM methodology using different Cu/O species to determine which best facilitated the dealkylation event. It was determined that radical recombination between a Cu(II)-oxyl and a substrate radical to form an unstable copper-alkoxide intermediate was best suited to carry out this reaction. Structure-function analysis was used to rationalize the electronic features which made a variety of diverse imino-oxy acetic acid analogues such unexpectedly good PAM substrates (104-5 M-1s-1). To observe the effect oxygen insertion and placement had on substrates between N-benzoylglycine and benzaldehyde imino-oxy acetic acid structures, PAM activity was correlated with NBO/MEP calculations on selected PHM-docked structures. This work concluded that the imino-oxy acetic acid was a favored substrate for PAM because its oxime electronically is very similar to the amide present in glycine-extended analogues.

The vast variety of long-chain fatty acid amides identified in biological systems is intriguing. The general structure of a fatty acid amide is R-CO-NH-X, where R is an alkyl group and X is derived from an immense variety of biogenic amines. Although structurally simple, the bioactivities of these molecules as signaling lipids are very diverse and have just recently begun to emerge in the literature. Interest in the long-chain fatty acid amides dramatically increased following the identification and characterization of one specific N-acylethanolamine, N-arachidonoylethanolamine (anandamide), as the endogenous ligand for the cannabinoid receptors in the mammalian brain. Since this discovery, the details of N-acylethanolamine metabolism have been elucidated. However, a lesser extent of progress has been made in the last twenty years to identify and study the non-N-acylethanolamine long-chain fatty acid amides. The focus of this dissertation is the elucidation of the biosynthetic pathways for long-chain fatty acid amides, including N-acylglycines, primary fatty acid amides, N-acylarylalkylamides, and N-acylethanolamines. The details of long-chain fatty acid amide metabolism will lead to the determination of possible therapeutic targets. We identified mammalian glycine N-acyltransferase like 3 as the enzyme that catalyzes the formation of long-chain N-acylglycines in mouse N18TG2 neuoblastoma cells, identified and quantified a panel of long-chain fatty acid amides in Drosophila melanogaster extracts by LC/QTOF-MS, established Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism, and identified arylalkylamine N-acyltransferase like 2 as the enzyme that catalyzes the formation of long-chain N-acylserotonins and N-acyldopamines in Drosophila melanogaster.

Primary fatty acid amides (PFAMs) and N-acylglycines (NAGs) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotor activity, angiogenesis, vasodilatation, gap junction communication, and many other processes. Oleamide is the best-studied of the PFAMs, while the in vivo activity of the others is largely unstudied. Even less is known about the NAGs, as their discovery as novel compounds is much more recent due to low endogenous levels. Herein is described extraction and quantification techniques for PFAMs and NAGs in cultured cells and media using solvent extraction combined with solid phase extraction (PFAM) or thin layer chromatography (NAG), followed by gas chromatography-mass spectroscopy to isolate and quantify these lipid metabolites. The assays were used to examine the endogenous amounts of a panel of PFAMs as well as the conversion of corresponding free fatty acids (FFAs) to PFAMs over time in several cell lines. The cell lines demonstrated the ability to convert all FFAs, including a non-natural FFA, and an ethanolamine to the corresponding PFAM. Different patterns of relative amounts of endogenous and FFA-derived PFAMs were observed in the cell lines tested. Essential to identifying therapeutic targets for the many disorders associated with PFAM signaling is understanding the mechanism(s) of PFAM and NAG biosynthesis. Enzyme expression studies were conducted to determine potential metabolic enzymes in the model cell lines in an attempt to understand the mechanism(s) of PFAM biosynthesis. It was found that two of the cell lines which show distinct metabolisms of PFAMs also demonstrate unique enzyme expression patterns, and candidate enzymes proposed to perform PFAM and NAG metabolism are described. RNAi knockdown studies revealed further information about the metabolism of PFAMs and calls into question the recently proposed involvement of cytochrome c. Isotopic labeling studies showed there are two pathways for PFAM formation. A novel enzyme is likely to be involved in formation of NAGs from acyl-CoA intermediates.