Mouse aminoacylase 3: a metalloenzyme activated by cobalt and nickel.
Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene. Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn(2+)-metalloenzyme aminoacylase 2 (AA2). A 3D model of mouse AA3 was created based on homology with AA2. The model showed a putative metal binding site formed by His21, Glu24 and His116, and Arg63, Asp68, Asn70, Arg71, Glu177 and Tyr287 potentially involved in catalysis/substrate binding. The mutation of each of these residues to alanine inactivated AA3 except Asn70 and Arg71, therefore the corrected 3D model of mouse AA3 was created. Wild type (wt) mouse AA3 expressed in E. coli contained approximately 0.35 zinc atoms per monomer. Incubation with Co(2+) and Ni(2+) activated wt-AA3. In the cobalt-activated AA3 zinc was replaced with cobalt. Metal removal completely inactivated wt-AA3, whereas addition of Zn(2+), Mn(2+) or Fe(2+) restored initial activity. Co(2+) and to a lesser extent Ni(2+) increased activity several times in comparison with intact wt-AA3. Co(2+) drastically increased the rate of deacetylation of Ac-DCVC and significantly increased the toxicity of Ac-DCVC in the HEK293T cells expressing wt-AA3. The results indicate that AA3 is a metalloenzyme significantly activated by Co(2+) and Ni(2+).
Department of Medicine, David Geffen School of Medicine, University of California at Los Angeles, CA 90095, USA.
Kirill
K
Tsirulnikov
Natalia
N
Abuladze
Debra
D
Newman
Sergey
S
Ryazantsev
Talya
T
Wolak
Nathaniel
N
Magilnick
Myong-Chul
MC
Koag
Ira
I
Kurtz
Alexander
A
Pushkin
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009532:Nickel / Q000494:pharmacology*; D011487:Protein Conformation
D003035:Cobalt; D009532:Nickel; D000581:Amidohydrolases; C483737:aminoacylase III, mouse