Tyrosine modification enhances metal-ion binding

Tyrosine sulfation is a common modification of many proteins, and the ability to phosphorylate tyrosine residues is an intrinsic property of many growth-factor receptors. In the present study, we have utilized the peptide hormone CCK₈ (cholecystokinin), which occurs naturally in both sulfated and unsulfated forms, as a model to investigate the effect of tyrosine modification on metal-ion binding. The changes in absorbance and fluorescence emission on Fe³⁺ binding indicated that tyrosine sulfation or phosphorylation increased the stoichiometry from 1 to 2, without greatly affecting the affinity (0.6–2.8 μM at pH 6.5). Measurement of Ca²⁺ binding with a Ca²⁺-selective electrode revealed that phosphorylated CCK₈ bound two Ca²⁺ ions. CCK₈ and sulfated CCK₈ each bound only one Ca²⁺ ion with lower affinity. Binding of Ca²⁺, Zn²⁺ or Bi³⁺ to phosphorylated CCK₈ did not cause any change in absorbance, but substantially increased the change in absorbance on subsequent addition of Fe³⁺. The results of the present study demonstrate that tyrosine modification may increase the affinity of metal-ion binding to peptides, and imply that metal ions may directly regulate many signalling pathways.