Mechanism of Peptide Amidation: Structural and Kinetic Studies

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Intellectual Merit: Amidated peptides are part of signaling systems in species ranging from Aplysia to humans. In all cases, glycine-extended intermediates are transformed into active amidated hormones by oxidative cleavage of the glycine N-C(alpha) bond by a bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes two domains that catalyze the two sequential reactions producing amidated peptide; alpha-hydroxylation of the glycine (PHM) and excision of the C(alpha)-N bond to give the alpha-amidated peptide plus glyoxylate (PAL). PHM contains two redox active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for hydroxylation of glycine-extended substrates. PAL is a zinc-containing lyase that cleaves the C(alpha)-N bond after hydroxylation. As part of previous work, the structures of reduced and oxidized PHM, alone and with substrates, were determined. Structural and kinetic characterization of small molecule ligands of PHM as well as selected mutants were also accomplished. In a very important development, the determination of the structure of the catalytic core of PAL was accomplished. This led to the determination of the structure of PAL in complex with a non-peptidic substrate (hyrdoxyhippuric acid), proposing a mechanism, and the design and validation of a PAL inhibitor. In addition, kinetic studies of selected mutants were carried out to confirm the mechanism. This project involves further characterization of the PHM copper centers using single crystal and solution XAFS, and identification of the species that abstracts the substrate hydrogen in PHM. Studies on PAL are centered on addressing additional features of its mechanism by studying mutants, the effects of reductants, and the kinetics of a new inhibitor. The structure of PAM will be determined as a complete construct containing the PHM and PAL domains and as a complex of the two domains. The interaction between the two domains will be characterized using solution methods. The project will also explore whether the two-step reaction takes place with release of the PHM product or if it occurs processively by an internal transfer from PHM to PAL. The studies will advance understanding of peptide amidation which is a fundamental step in signal transduction. They will, in addition, provide an outstanding paradigm for understanding long range electron transfers as well as processivity in two step enzymes. Moreover, PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase -the enzyme that converts dopamine to norepinephrine, a key neurotransmitter. Broader Impact The PI is actively involved in bringing the excitement of science to high school students, undergraduates and underrepresented minorities. He teaches classes in two undergraduate courses, and has collaborations with two laboratories at undergraduate teaching institutions. He participated in a broad range of outreach activities that have been directed at members of underrepresented groups. The laboratory of the PI participates annually in the Department-wide Science Day organized in conjunction with the Baltimore City school system for students from the inner city. The PI has a strong presence in the Hispanic scientific community and in Latin American science. He is a founding member and past president of the Society for Latin American Biophysicists (SOBLA). The PI currently trains four minority students in his laboratory.