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J. Biol. Chem., Vol. 282, Issue 48, 34684-34692, November 30, 2007

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Inhibition of Human Dimethylarginine Dimethylaminohydrolase-1 by S-Nitroso-L-homocysteine and Hydrogen Peroxide

ANALYSIS, QUANTIFICATION, AND IMPLICATIONS FOR HYPERHOMOCYSTEINEMIA^*

From the Division of Medicinal Chemistry, College of Pharmacy, and the Texas Institute for Drug and Diagnostic Development, University of Texas, Austin, Texas 78712

The plasma concentrations of two cardiovascular risk factors, total homocysteine (tHcy) and asymmetric dimethylarginine (ADMA), correlate with decreased levels of endothelium-derived nitric oxide and subsequent endothelial dysfunction. Homocysteine has been proposed to inhibit the catabolic enzyme of ADMA, dimethylarginine dimethylaminohydrolase (DDAH), but the mechanism of this inhibition has not been fully elucidated. Here, the human DDAH isoform-1 (DDAH-1) is heterologously expressed and purified. Cys²⁷⁴ and His¹⁷³ are identified as active site residues and the pH rate dependence is described. Because oxidation of the active site Cys has been suggested as an inhibitory mechanism in patients with hyperhomocysteinemia, the sensitivity of DDAH-1 to inhibition by L-homocysteine, H₂O₂, and S-nitroso-L-homocysteine is quantified. DDAH-1 is surprisingly insensitive to inactivation by the powerful oxidant, H₂O₂ (0.088 M^–1 s^–1), possibly because of a substrate-assisted mechanism that allows the active site cysteine to remain predominantly protonated and less reactive in the resting enzyme. In contrast, DDAH-1 is sensitive to inactivation by S-nitroso-L-homocysteine (3.79 M^–1 s^–1). This work illustrates how a particular catalytic mechanism can result in selective redox regulation and has possible implications for hyperhomocysteinemia.

Received for publication, August 28, 2007

^* This work was supported in part by Grant RSG-05-061-01-GMC from the American Cancer Society, Grant F-1572 from the Robert A. Welch Foundation, and a seed grant from the Texas Institute for Drug and Diagnostic Development at the University of Texas, Austin. 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 To whom correspondence should be addressed: 1 University Station, A1935, PHAR-MED CHEM, The University of Texas, Austin, TX 78712. Tel.: 512-232-4000; Fax: 512-232-2606; E-mail: WaltFast{at}mail.utexas.edu.

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