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J. Biol. Chem., Vol. 281, Issue 30, 20873-20882, July 28, 2006

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Role for an Essential Tyrosine in Peptide Amidation^*

From the Neuroscience Department, University of Connecticut Health Center, Farmington, Connecticut 06030-3401

The catalytic core of the peptidyl--hydroxyglycine -amidating lyase (PAL) domain of peptidylglycine -amidating monooxygenase was investigated with respect to its ability to function as a ureidoglycolate lyase and the identity and role of its bound metal ions. The purified PAL catalytic core (PALcc) contains molar equivalents of calcium and zinc along with substoichiometric amounts of iron and functions as a ureidoglycolate lyase. Limiting iron availability in the cells synthesizing PALcc reduces the specific activity of the enzyme produced. Concentrated samples of native PALcc have an absorption maximum at 560 nm, suggestive of a phenolate-Fe(III) charge transfer complex. An essential role for a Tyr residue was confirmed by elimination of PAL activity following site-directed mutagenesis. Purified PALcc in which the only conserved Tyr residue (Tyr⁶⁵⁴) was mutated to Phe was secreted normally, but was catalytically inactive and lacked bound iron and bound zinc. Our data demonstrate an essential role for Tyr⁶⁵⁴ and suggest that it serves as an Fe(III) ligand in an essential iron-zinc bimetallic site.

Received for publication, December 29, 2005 , and in revised form, May 15, 2006.

^* This work was supported by National Institutes of Health Grants DK32949 (to B. A. E. and R. E. M.) and NS27583 (to N. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Pediatrics, University of Medicine & Dentistry of New Jersey-Robert Wood Medical School, New Brunswick, NJ 08903-0019.

² Present address: Dept. of Environmental and Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University, Beaverton, OR 97006-8921.

³ To whom correspondence should be addressed: Dept. of Neuroscience, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3401. Tel.: 860-679-8898; Fax: 860-679-1885; E-mail: eipper{at}uchc.edu.

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