Inhibition of human dimethylarginine dimethylaminohydrolase-1 by S-nitroso-L-homocysteine and hydrogen peroxide. Analysis, quantification, and implications for hyperhomocysteinemia.
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.
Hong
Lin
L
Division of Medicinal Chemistry, College of Pharmacy, and Texas Institute for Drug and Diagnostic Development, University of Texas, Austin, Texas 78712, USA.
Fast
Walter
W
D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D020138:Hyperhomocysteinemia / Q000188:drug therapy* / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D006710:Homocysteine; C079202:S-nitrosohomocysteine; D006861:Hydrogen Peroxide; D000581:Amidohydrolases; C059999:dimethylargininase; D003545:Cysteine