Degree Granting Department
David Merkler, Ph.D.
Hydroxylation, Amidation, Imino-oxy, Acylglycine
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 °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 Cu[subscript]M. 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.
Scholar Commons Citation
McIntyre, Neil R., "Mechanistic studies of peptidylglycine alpha-amidating monooxygenase (PAM)" (2008). Graduate Theses and Dissertations.