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

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.Endothelium-derived nitric oxide (NO) regulates vasorelaxation as well as other vascular functions (1). Endothelial dysfunction is observed in many conditions including hypercholesterolemia, hypertension, diabetes, hyperhomocysteinemia, chronic renal failure, smoking, and aging, and often correlates with a decrease in endothelium-derived NO and the associated vasodilation (2). Elevated plasma concentrations of at least two cardiovascular risk factors, asymmetric N ,N -dimethyl-L-arginine (ADMA) 2 and total homocysteine (tHcy), are known to be associated with endothelial dysfunction (3-5). These two markers have been proposed to be mechanistically linked and to mediate at least part of the pathobiology of patients with hyperhomocysteinemia (6 -9). Much of the pathway for inhibition of NO synthesis by ADMA is known. This endogenously produced arginine analog can be transported by the y ϩ cationic amino acid transporter (10) and is known to be an inhibitor of all three NO synthase isoforms (11). Pathological accumulation of ADMA has been proposed as a ubiquitous mechanism for endothelial dysfunction and has even been called an "über marker" for adverse cardiovascular conditions (12). The plasma concentrations of ADMA are partially controlled by one or both isozymes of the enzyme dimethylarginine dimethylaminohydrolase (DDAH-1 and DDAH-2), which hydrolyze ADMA (Scheme 1, structure 1) to the non-inhibitory (or less-inhibitory) products citrulline (Scheme 1, structure 2) and dimethylamine (Scheme 1, structure 3) (11). The systemic inhibition of DDAH activity by small molecules (13) and the transgenic overexpression of the DDAH-1 isoform (14) result in respective increases or decre...

Protection of DDAH2 Overexpression Against Homocysteine-Induced Impairments of DDAH/ADMA/NOS/NO Pathway in Endothelial Cells

“…Similar findings have been made by other investigators in hearts of dogs with congestive heart failure [26] and in lungs of rats with pulmonary hypertension [27]. Homocysteineinduced oxidation stress might be implicated in the suppression of DDAH transcription or expression, because DDAH is a redox-sensitive enzyme resulting from a critical sulfhydryl group within its catalytic site [16]. Taken together, these results indicate that suppressed expression of DDAH2 rather than DDAH1 contributes to inhibition of DDAH activity and NO synthesis induced by homocysteine in endothelial cells.…”

“…It is intriguing that DDAH activity was significantly inhibited after homocysteine treatment in endothelial cells, but DDAH protein expression remained unchanged [15]. Although homocysteine was found to inhibit the activity of DDAH directly [16], overexpression of DDAH1 could not protect the mice with dietary hyperhomocysteinemia from endothelial dysfunction [17]. These results allowed authors to speculate that DDAH2 rather than DDAH1 might play a pivotal role in ADMA metabolism of endothelial cells and homocysteine-induced endothelial dysfunction.…”

Contribution of Endogenous Inhibitor of Nitric Oxide Synthase to Hepatic Mitochondrial Dysfunction in Streptozotocin-induced Diabetic Rats

“…Because DDAH is the major enzyme for ADMA degradation and its high expression in liver [18]. Furthermore, DDAH can be redox-regulated [30], and decreased DDAH activity as well as enhanced oxidative stress were found in the liver of diabetic rats in the present study. Consistent results were also observed in the aortas of diabetic rats [16].…”

Role of Dimethylarginine Dimethylaminohydrolases in the Regulation of Endothelial Nitric Oxide Production

“…However, in order to assess the relative contribution of each enzyme, a detailed analysis of the enzyme kinetics of each isoform is necessary. Unfortunately, detailed biochemical studies have only been published for DDAH-1 (19,42 (19,42). In regard to DDAH-2, previous attempts at purifying the protein have been unsuccessful primarily due to solubility issues with recombinant enzyme.…”