Molecular mechanisms for antibody-mediated modulation of peptide-displaying enzyme sensors

The generation of molecular sensors based on peptide-displaying enzymes for the detection of antibodies or antigens represents an innovative field of protein engineering The knowledge of the underlying molecular mechanisms of enzymatic modulation in such sensors would be of great importance for the rational construction and improvement of responsiveness of new peptide-enzy me molecules. Here we analyze the enzymatic characteristics of three differ ent kinds of sensors based in engineered beta-galactosidase, alkaline phosp hatase and beta-lactamase, to explore a common activation basis. We describ e two different categories of enzyme sensors, In one of them, including onl y some modified beta-lactamases, the enzymatic activity is inhibited upon l igand binding and it seems to be caused by the steric coverage of the activ e site by the bound antibody. In a second group, embracing members of the t hree studied enzymes, the ability to be modulated upon effector binding dep ends on the ratio between the k(cat) of the engineered enzyme and the k(cat ) of the intact enzyme, This proves a common mechanism for enzymatic modula tion of enzyme biosensors that is probably caused by conformational effects induced by the bound antibody on the enzyme, (C) 2000 Academic Press.