Mechanism of Peptide Amidation: Structural and Kinetic Studies

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Amidated peptides are fundamental signaling molecules found in species ranging from Aplysia to humans. Surprisingly, peptide amidation is not carried out by a transamination reaction, but by oxidative cleavage, glycine-extended precursors. A bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes this reaction. The PAM gene encodes two domains (PHM and PAL) that catalyze two sequential reactions: alpha-hydroxylation of the glycine (PHM) and excision of the C alpha-N bond to give alpha-amidated peptide (PAL). PHM contains two redox active copper atoms that, upon reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. PAL is a zinc containing lyase that cleaves the C alpha-N bond after hydroxylation of the C alpha. This project will employ X-ray diffraction, kinetic experiments, peptide design, and site directed mutagenesis to elucidate important aspects of the mechanism of reactions catalyzed by PHM and PAL. Understanding the chemistry of PAM, especially that of the first step of the reaction, will not only contribute to the field of peptide amidation, but will also provide an outstanding paradigm for understanding long range electron transfer and the prevention of production of deleterious oxygen species. Broader Impacts: The PI of this project is actively involved in teaching as well as in bringing the excitement of science to underrepresented minorities. Mentoring of minority students will be achieved through participation in the Meyerhoff Scholars Programs of the University of Maryland Baltimore Campus. The project will also involve participation in the Science Day organized in conjunction with the Baltimore City school system.