This study describes the development of a universal phosphorylated peptide-binding protein designed to simultaneously detect serine, threonine and tyrosine kinases. The Escherichia coli alkaline phosphatase (EAP) is a well-defined nonspecific phosphated monoesterase and Ser-, Thr- or Tyr-phosphorylated peptides served as substrates for EAP in preliminary experiments. Based on the known catalytic mechanism of EAP, the recombinant site-directed mutant EAP-S102L was generated, whose catalytic activity was blocked, but its binding ability was preserved. For EAP-S102L the catalytic rate constant, k(cat), was reduced by a factor of 1000, while the Michaelis-Menten constant, K(m), remained almost unchanged. Crystallographic analysis of the EAP-S102L/phophorylated peptide complex revealed that EAP-S102L could bind the phosphate group of the phosphorylated peptide but lacked nucleophilic attack potential which was essential for the catalytic ability of EAP. Finally, by combining the fluorescence-labeled EAP-S102L with non-phophorylated peptide chips, kinases could be detected from tumor cell samples. The recombinant EAP-S102L construct is perhaps the first functional binding protein derived from a native enzyme, illustrating how one single mutation tremendously alters protein function.