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J. Biol. Chem., Vol. 283, Issue 18, 12188-12201, May 2, 2008

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Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of Activity^*

John E. Dominy, Jr.¹, Jesse Hwang, Stephanie Guo, Lawrence L. Hirschberger, Sheng Zhang, and Martha H. Stipanuk²

From the Department of Nutritional Sciences and the Cornell University Life Sciences Core Laboratories Center, Cornell University, Ithaca, New York 14853

Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the -sulfur of residue cysteine 93 and the aromatic side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examined what effect cofactor formation had on activity in vitro. Like other cross-linked cofactor-containing enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe²⁺) and O₂. Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiologically relevant cysteine concentrations, cofactor formation increased CDO catalytic efficiency by 10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiological consequences.

Received for publication, January 2, 2008 , and in revised form, February 20, 2008.

^* This work was supported in part by NIDDK Grant PHS DK056649 from the National Institutes of Health (to M. H. S.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Supported by a graduate student fellowship from the National Science Foundation.

² To whom correspondence should be addressed: Division of Nutritional Sciences, 227 Savage Hall, Cornell University, Ithaca, NY 14853. Tel.: 607-255-2638; Fax: 607-255-1033; E-mail: mhs6{at}cornell.edu.

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