title: Structural and functional characterization of the Zn(II) site in dimethylargininase-1 (DDAH-1) from bovine brain. Zn(II) release activates DDAH-1.
abstract: L-N(omega),N(omega)-dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Zn(II)-containing enzyme that, through hydrolysis of side-chain methylated l-arginines, regulates the activity of nitric-oxide synthase. Herein we report the structural and functional properties of the Zn(II)-binding site in DDAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzyme. Native DDAH-1 could be fully or partially activated using various concentrations of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme by the bulky complexing agents EDTA and 1,10-phenantroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The apparent Zn(II)-dissociation constant of 4.2 nm, determined by 19F NMR using the chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid), lies in the range of intracellular free Zn(II) concentrations. These results suggest a regulatory role for the Zn(II)-binding site. The coordination environment of the Zn(II) in DDAH-1 has been examined by Zn K-edge x-ray absorption spectroscopy. The extended x-ray absorption fine structure observed is consistent with Zn(II) being coordinated by 2 S and 2 N (or O) atoms. The biological implications of these findings are discussed.
authors.affiliation: Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Knipp
authors.forename: J M
authors.initials: JM
authors.lastname: Charnock
authors.forename: C D
authors.initials: CD
authors.lastname: Garner
authors.forename: M
authors.initials: M
authors.lastname: Vasák
mesh_terms: D000581:Amidohydrolases; D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D004789:Enzyme Activation; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
chemical_list: D006867:Hydrolases; D000581:Amidohydrolases; C059999:dimethylargininase; D015032:Zinc