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Ligand binding and allostery can emerge simultaneously

A heterotropic allosteric effect involves an effector molecule that is distinct from the substrate or ligand of the protein. How heterotropic allostery originates is an unanswered question. We have previously created several heterotropic allosteric enzymes by recombining the genes for TEM1 β‐lactamase (BLA) and maltose binding protein (MBP) to create BLAs that are positively or negatively regulated by maltose. We show here that one of these engineered enzymes has ∼106 M−1 affinity for Zn2+, a property that neither of the parental proteins possesses. Furthermore, Zn2+ is a negative effector that noncompetitively switches off β‐lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn2+‐binding site does not involve a histidine or a cysteine, which is atypical of natural Zn2+‐binding sites. These studies also implicate helices 1 and 12 of the BLA domain in allosteric signal propagation. These results support a model for the evolution of heterotropic allostery in which effector affinity and allosteric signaling emerge simultaneously.