Inactivation of two diverse enzymes in the amidinotransferase superfamily by 2-chloroacetamidine: dimethylargininase and peptidylarginine deiminase.
The enzymes dimethylargininase [dimethylarginine dimethylaminohydrolase (DDAH); EC 3.5.3.18] and peptidylarginine deiminase (PAD; EC 3.5.3.15) catalyze hydrolysis of substituted arginines. Due to their role in normal physiology and pathophysiology, both enzymes have been identified as potential drug targets, but few useful inhibitors have been reported. Here, we find that 2-chloroacetamidine irreversibly inhibits both DDAH from Pseudomonas aeruginosa and human PAD4 in a time- and concentration-dependent manner, despite the nonoverlapping substrate specificities and low levels of amino acid identity of their catalytic domains. Substrate protection experiments indicate that inactivation occurs by modification at the active site, albeit with modest affinity. Mass spectral analysis demonstrates that irreversible inactivation of DDAH occurs through selective formation of a covalent thioether bond with the active-site Cys249 residue. The mechanism of inactivation by 2-chloroacetamidine is analogous to that of chloromethyl ketones, a set of inhibitors that have found wide application because of their specific covalent modification of active-site residues in serine and cysteine proteases. Likewise, 2-chloroacetamidine may potentially find wide applicability as a general pharmacophore useful in delineating characteristics of the amidinotransferase superfamily.
Stone
Everett M
EM
Graduate Program in Cell and Molecular Biology, The University of Texas, Austin, Texas 78712, USA.
Schaller
Terezie H
TH
Bianchi
Helena
H
Person
Maria D
MD
Fast
Walter
W
D000578:Amidines / Q000494:pharmacology*; D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D000076342:Protein-Arginine Deiminases
C509120:2-chloroacetamidine; D000578:Amidines; D017931:DNA Primers; D004791:Enzyme Inhibitors; D006867:Hydrolases; D000581:Amidohydrolases; D000076342:Protein-Arginine Deiminases; C059999:dimethylargininase