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

Lin Hong,Walter Fast

doi:10.1074/jbc.m707231200

Abstract

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. Cys(274) and His(173) 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, H(2)O(2), and S-nitroso-L-homocysteine is quantified. DDAH-1 is surprisingly insensitive to inactivation by the powerful oxidant, H(2)O(2) (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.

Highlights

Endothelium-derived nitric oxide (NO) regulates vasorelaxation as well as other vascular functions [1]
The results clearly show that human dimethylarginine dimethylaminohydrolase (DDAH)-1 is not sensitive to inhibition by physiological concentrations of H2O2, despite having an active site cysteine nucleophile
Characterization of DDAH isoform-1 (DDAH-1) by electrospray ionization (ESI)-MS showed a major peak at 33435 Ϯ 10 Da which matches the mass calculated from the amino acid sequence of His6tagged DDAH-1 after removal of the N-terminal methionine residue (33441 Da)

Summary

EXPERIMENTAL PROCEDURES

Cloning and Purification of Human DDAH-1—The coding region for human DDAH-1 (DDAH-1, Protein GI: 6831528) was amplified from an I.M.A.G.E. clone (5189970) obtained from the American Type Culture Collection (ATCC 7273181, Manassas, VA) using two specific end primers: 5Ј-CTAGCTAGCATGGCCGGGCTCGGCCACCCC-3Ј and 5Ј-CGGGATCCTCAGGAGTCTACTTTCTTG-3Ј. At various time points between 0 and 20 min, aliquots (10 ␮l) of the preincubation mix were diluted 20-fold into a reaction mixture containing ADMA (0.1 mM) and 60 units of bovine liver catalase, all in K2HPO4 buffer (100 mM) at pH 7.5. To monitor the time-dependent inactivation of DDAH-1 by HcyNO, enzyme (40 ␮M) was incubated with varying concentrations of freshly prepared HcyNO (0.2– 4 mM) in K2HPO4 buffer (100 mM) at pH 7.5. Subsequent addition of DTT (25 mM) or glutathione (25 mM) to this treated preincubation mix was followed by 20-fold dilution of aliquots (10 ␮l) at various timepoints (0 –20 min) into a reaction mixture containing ADMA (0.1 mM) in K2HPO4 buffer (100 mM) at pH 7.5, which was assayed for remaining DDAH-1 activity as described above. The preincubation solution was treated with DTT (5 mM) and recovery of activity was monitored as described above

RESULTS

39 Ϯ 9 44 Ϯ 1

DISCUSSION