Zinc binding in bovine alpha-lactalbumin: sequence homology may not be a predictor of subtle functional features

alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution.