Properties of a 19-kDa Zn2+-binding protein and sequence of the Zn2+-binding domains

A novel 19-kDa protein has been described recently [Brand, I. A. and Soling, H.-D. (1986) J. Biol. Chem. 261, 5895 – 5900] which is able to inactivate 6-phosphofructo-1-kinase reversibly in a Zn2+-dependent manner. We present now additional biochemical and physicochemical data concerning this protein. It is extremely acidic with 40% glutamic and 15% aspartic acid residues. It contains no sulfur, aromatic amino acids, histidine or isoleucine. The protein has four binding sites for Zn2+ with an apparent Kd of about 6 μM. Two of these binding sites are called unspecific as Zn2+ is displaced from these binding sites at physiological concentrations of free Mg2+ (0.75 mM) and at high salt concentrations (100 mM NaCl). Whereas Mg2+-binding to the two other so-called specific Zn2+-binding sites occurs only at Mg2+ concentrations at about 5 mM. The four Zn2+-binding sites were detected on a tryptic peptide (T8) of 43 amino acid residues, which still possessed biological activity. This peptide has been sequenced and is characterized by four clusters of acidic amino acids separated by only a few neutral amino acids. The two specific Zn2+-binding sites could be detected in the C-terminal portion of T8, the two unspecific Zn2+-binding sites must therefore be located at the N-terminal portion. The Zn2+-binding domains of the 19-kDa Zn2+-binding protein described here are completely different from those of the ‘zinc finger’ discovered in several DNA-binding proteins.