Peptidylglycine-R-Hydroxylating Monooxygenase Generates Two Hydroxylated Products from Its Mechanism-Based Suicide Substrate, 4-Phenyl-3-butenoic Acid ² William J. Driscoll,* ,‡ Simone Ko ¨nig, § Henry M. Fales, § Lewis K. Pannell, | Betty A. Eipper, ⊥ and Gregory P. Mueller ‡ Department of Physiology, F. Edward He ´ bert School of Medicine, Uniformed SerVices UniVersity of the Health Sciences, Bethesda, Maryland 20814, Laboratory of Biophysical Chemistry, National Heart Lung and Blood Institute, and Laboratory of Bioorganic Chemistry, National Institute of Diabetes and DigestiVe and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, and Department of Neuroscience, Johns Hopkins UniVersity School of Medicine, Baltimore, Maryland 21205 ReceiVed February 2, 2000; ReVised Manuscript ReceiVed April 21, 2000 ABSTRACT: The bifunctional enzyme peptidylglycine-R-amidating monooxygenase mediates the conversion of C-terminal glycine-extended peptides to their active R-amidated products. Peptidylglycine-R- hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the first reaction in this two-step process. The olefinic compound 4-phenyl-3-butenoic acid (PBA) is the most potent irreversible, mechanism-based PHM inactivator known. While the details of the inhibitory action of PBA on PHM remain undefined, covalent modification of the protein has been proposed as the underlying mechanism. We report here that, in the process of inactivating PHM, PBA itself serves as a substrate without covalently labeling the enzyme. Approximately 100 molecules of PBA are metabolized per molecule of PHM inactivated, under saturating conditions. The metabolism of PBA by PHM generates two hydroxylated products, 2-hydroxy- 4-phenyl-3-butenoic acid and its allylic isomer, 4-hydroxy-4-phenyl-2-butenoic acid. While one enantiomer for each product is significantly favored in the reaction, both are produced. From these observations, we conclude that hydroxylated PBA products are formed by a delocalized free radical mechanism and that the lack of absolute stereospecificity indicates significant freedom of movement within the catalytic site. The ability of PHM to metabolize PBA suggests that the physiological functions of PHM may include the hydroxylation of substrates other than those containing terminal glycines. Intercellular peptide messengers regulate physiological mechanisms essential for life. More than half of all known neuroendocrine peptides are R-amidated at their carboxy- termini, and in most cases this structural feature is requisite for receptor recognition and signal transduction (1-3). R-Amidation is catalyzed by peptidylglycine-R-amidating monooxygenase (PAM), 1 which constitutes the only known mechanism for generating R-amidated peptides in vivo. PAM is a bifunctional enzyme consisting of peptidylglycine-R- hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidyl-R-hydroxyglycine R-amidating lyase (PAL, EC 4.3.2.5). The hydroxylase and lyase domains of PAM sequentially catalyze the two steps required for converting glycine-extended peptide precursors to active peptide amides (1-3). PHM is rate-limiting in this sequence and thus represents a key control point in the bioactivation of peptide messengers. The PHM and PAL domains of PAM may be separated with full retention of their respective enzymatic activities (4-6). This feature has facilitated the study of each catalytic domain. To date, attention has focused on PHM because of its regulatory role in controlling R-amidation and its homology to the norepinephrine synthesizing enzyme, dopamine -monooxygenase (DBM, EC 1.14.17.1) (7,8). The crystallographic structure of PHM has provided considerable insight into the mechanism by which PHM hydroxylates its substrates (9). PHM is composed of 2 domains that are approximately equal in size (∼150 residues). Each domain coordinates one copper atom on opposite sides of a solvent-accessible catalytic cleft. This open coordination positions the two copper atoms (CuA and CuB) 11 Å apart and, thus, precludes the formation of a typical binuclear center in which oxygen bridges the two metal ions. Substrate- mediated electron transfer between the metal ions (10) and channeling of superoxide (11) have been proposed to play a ² This work was supported by NIH Grant NS-34173 (G.P.M.), USUHS Grant RO7644 (G.P.M.), and NIH Grant DK-32949 (B.A.E). * To whom correspondence should be addressed at the Department of Physiology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799. Tel.: 301-295- 3515; Fax: 301-295-3566; e-mail: wdriscoll@USUHS.mil. ‡ Department of Physiology, Uniformed Services University of the Health Sciences. § Laboratory of Biophysical Chemistry, National Heart Lung and Blood Institute. | Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases. ⊥ Department of Neuroscience, Johns Hopkins University School of Medicine. 1 Abbreviations: PAM, peptidylglycine-R-amidating monooxyge- nase; PHM, peptidylglycine-R-hydroxylating monooxygenase; PAL, peptidyl-R-hydroxyglycine R-amidating lyase; DBM, dopamine -mo- nooxygenase; PBA, 4-phenyl-3-butenoic acid; 2-OH-PBA, 2-hydroxy- 4-phenyl-3-butenoic acid; 4-OH-PBA, 4-hydroxy-4-phenyl-2-butenoic acid; NMR, nuclear magnetic resonance; LC/MS, liquid chromatog- raphy/mass spectrometry; HPLC, high-performance liquid chromatog- raphy; CID, collision-induced dissociation; MES, 2-(N-morpholino)- ethanesulfonic acid; TFA, trifluoroacetic acid; SDS, sodium dodecyl sulfate; PAGE, poylacrylamide gel electrophoresis. 8007 Biochemistry 2000, 39, 8007-8016 10.1021/bi0002380 CCC: $19.00 © 2000 American Chemical Society Published on Web 06/08/2000