Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamination reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-Ca bond. In higher organisms, the bifunctional enzyme peptidylglycine a-amidating monooxygenase (PAM) catalyzes this reaction. The PAM gene encodes two domains (PHM and PAL) that when separated, either through cleavage or through independent expression, retain their individual enzymatic activities. Together they catalyze the two sequential reactions that produce amidated peptide: a-hydroxylation of the glycine (PHM) and excision of the Ca-N bond to give a-amidated peptide product and glyoxylate (PAL). PHM contains two redox active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. PAL is zinc containing lyase that cleaves the Ca-N bond after hydroxylation of the Ca. This project will use of x-ray diffraction and kinetic techniques to address questions about the origin of the broad substrate specificity of PHM and PAL, identify residues critical for PHM and PAL activity, and propose enzymatic mechanisms for both enzymes.

Peptide amidation is a fundamental biological process. Amidated peptides have been found in the signaling systems of species ranging from Aplysia to humans. The biosynthetic path used for this reaction allows the organism to strictly regulate the production of these peptides. Understanding the chemistry of this reaction, especially that of the first step, will not only contribute to the field of peptide amidation, but will also provide an outstanding paradigm for understanding long range electron transfers and the prevention of production of deleterious oxygen species.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Kamal Shukla
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Johns Hopkins University
United States
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