Site-specific substitution of glutamate for aspartate at position 59 of rat oncomodulin

Replacement of the aspartate residue at position 59 of rat oncomodulin by glutamate by oligonucleotide-directed mutagenesis has afforded a protein which more closely resembles rat parvalbumin, at least judged by its interaction with the luminescent lanthanide ion Eu³⁺. The single-peak ⁷F₀ → ⁵D₀ spectrum observed at pH 5.0 with the fully bound wild-type protein is replaced by one which clearly shows two features at 5791 and 5796 Å, arising from Eu³⁺ ions bound at the CD and EF sites, respectively. Furthermore, the pH dependence of the spectrum is substantially altered; the pK(a) observed for the CD domain, in which aspartate 59 residues, is shifted upward from pH 6.0 of the wild-type recombinant protein to pH 6.8 in the D59E mutant. Moreover, the maximum in the high-pH spectrum is shifted from 5781 to 5784 Å. All three changes are indicative of a CD binding domain having increased parvalbumin-like character. Interestingly, however, the D59E substitution has only a modest effect on the Ca²⁺ and Mg²⁺-binding properties of the CD domain. For the wild-type protein, K(Ca) = 7.8 x 10^-7 M and K(Mg) = 3 x 10^-3 M. These affinities are more than an order of magnitude weaker than those seen for various parvalbumins and substantiate previous claims for calcium specificity made for the oncomodulin CD domain. Replacement of aspartate 59 by glutamate resulted in minor increases in affinity of the CD domain for Ca²⁺ (K(Ca) = 5.5 x 10^-7 M) and Mg²⁺ (K(Mg) = 1 x 10^-3 M). These findings strongly suggest that residues in oncomodulin besides aspartate 59 are important determinants of the observed calcium specificity of the CD calcium-binding domain. The consequences of the substitution at residue 59 appear to be confined to the CD domain. For the EF site in wild-type recombinant oncomodulin, K(Ca) = 4.2 x 10^-8 M and K(Mg) = 1.6 x 10^-4 M. The corresponding values for the D59E site-specific variant are identical within experimental error (K(Ca) = 4.2 x 10^-8 M and K(Mg) = 1.8 x 10^-4 M).