BIOCHEMISTRY AND PHYSIOLOGY OF PEPTIDE AMIDATION

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

Studies will focus on peptidylglycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme whose catalytic domains (PHM and PAL) catalyze the two-step conversion of peptidylglycine substrates into alpha-amidated products. By investigating the catalytic, routing and cleavage domains of PAM, we hope to better understand how neuroendocrine peptides are produced and stored in regulated secretory granules. In Aim 1, key features in the catalytic cores of PHM and PAL will be identified by drawing on homologies to the corresponding Drosophila enzymes and dopamine beta- monooxygenase. The role of copper and disulfides in the folding of PHM will be investigated. The hypothesis that PHM consists of NH2- and COOH-terminal segments separated by a linker region will be tested. X-ray crystallographic studies of PHMcc will be completed. The COOH-terminus of PAL will be identified as will the Zn2+ binding site(s). Aim 2 is to determine whether all of the amidation activity in tissue extracts and serum can be attributed to PAM. Aim 3 focusses on the role(s) of the non-catalytic domains of PAM. The sites at which membrane PAM is cleaved will be identified and the fate of the resultant transmembrane/cytosolic domain fragment will be determined. We will test the hypothesis that membrane PAM subserves a signal transduction function in that interactions initiated in the cytosolic domain affect the properties of luminal PHM and PAL. Aim 4 is to define the role of phosphorylation in regulating PAM activity by identifying the cellular sites of phosphorylation, developing phosphorylation state specific antisera and mutating identified phosphorylation sites. P-CIP2 (PAM-COOH-terminal Interactor Protein) is a novel putative protein kinase identified in a yeast two hybrid screen by virtue of its ability to interact with the cytosolic domain of membrane PAM. Aim 5 is to define the manner in which P-CIP2 interacts with membrane PAM to affect its function. Peptides are widely employed as growth factors, and a better understanding of the processes involved in their biosynthesis will afford opportunities for controlling their production