Structural and functional characterization of the Zn(II) site in dimethylargininase-1 (DDAH-1) from bovine brain - Zn(II) release activates DDAH-1

L-N-omega,N-omega-Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Z n(II)-containing enzyme that, through hydrolysis of side-chain methylated L -arginines, regulates the activity of nitric-oxide synthase. Herein we repo rt the structural and functional properties of the Zn(II)-binding site in D DAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzym e. Native DDAH-1 could be fully or partially activated using various concen trations of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme b y the bulky complexing agents EDTA and 1,10-phenantroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The appa rent Zn(II)-dissociation constant of 4.2 nM, determined by F-19 NMR using t he chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetr aacetic acid), lies in the range of intracellular free Zn(II) concentration s. 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 stru cture 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.