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 n ever been measured in solution for human alpha-LA. This residue was genetic ally substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native (a lpha-LA by fluorescence methods, Surprisingly, desMetE49A (alpha-LA and the native bovine protein had similar affinities for both Zn2+ and Ca2+. Genet ic substitution of other possible candidates for Zn2+ chelating residues, w hich 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 st rong Zn2+ binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 3 7, which would participate in strong Zn2+ binding based on their propinquit y 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, su ggesting that the site identified from the X-ray structure did not correspo nd to strong zinc binding in solution. (C) 2000 Wiley-Liss, Inc.