The 2.15Å Structure of Peptidylglycine a-Hydroxylating Monooxygenase by MAD at the CuK Edge. S. T. Prigge, A. S. Kolhekar, B. A. Eipper and L. M. Amzel, Departments of Biophysics and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205. (Sponsored by L. M. Amzel)

Approximately 50% of mammalian bioactive hormones, neurotransmitters, and growth factors are peptides with a COOH-terminal carboxamide, generated by N-oxidative cleavage of a glycine-extended prohormone. A single bifunctional enzyme is responsible for catalyzing the a-amidation of these physiological regulators, many of which are inactive without a-amidation. Peptidylglycine a-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes two reactions at two separable catalytic domains: the first domain, peptidylglycine a-hydroxylating monooxygenase (PHM), catalyzes the copper, ascorbate, and molecular oxygen dependent a-hydroxylation of peptidylglycine substrates; the second domain, peptidyl-a-hydroxyglycine a-amidating lyase (PAL), is required to generate a-amidated peptide product and glyoxylate. The catalytic core of the PHM domain (residues 42-356 of rat PAM) has been crystallized in the orthorhombic space group P2₁2₁2₁ with cell dimensions a=68Å, b=70Å, c=81Å, and a=[beta]=[gamma]=90[ring]. Multi-wavelength anomalous dispersion (MAD) data has been collected around the copper absorption edge at the National Synchrotron Light Source and the Stanford Synchrotron Radiation Laboratory. The three dimensional structure of the PHM domain has been built and refined to 2.15Å with an R-factor of 19.5% and a cross validated R-factor of 27.9%. PHM is composed of two all-beta domains, both of which bind one of the active site copper atoms.