Two phosphoribosyltransferases in the purine salvage pathway exhibit exquisite substrate specificity despite the chemical similarity of their distinct substrates, but the basis for this discrimination was not fully understood. Ozeir et al. now employ a complementary biochemical, structural, and computational approach to deduce the chemical constraints governing binding and propose a distinct mechanism for catalysis in one of these enzymes, adenine phosphoribosyltransferase. These insights, built on data from an unexpected finding, finally provide direct answers to key questions regarding these enzymes and substrate recognition more generally.
