Incorporation of an artificial receptor into a native protein: New strategy for the design of semisynthetic enzymes with allosteric properties

The sugar-facilitated structure and enzymatic activity change of engineered myoglobins bearing a phenylboronic acid moiety, which were semisynthesized by a cofactor reconstitution method, were studied by the denaturation experiment, spectrophotometric titration of the pK(a) shift of the axial H₂O, circular dichloism (CD), and the kinetics of the myoglobin-catalyzed-aniline hydroxylation reaction. Both boronophenylalanine-appended myoglobin [Mb(m-Bphe)₂] and phenylboronic acid-appended myoglobin [Mb(PhBOH)₂] were stabilized by approximately 2 kcal/mol upon complexation with D-fructose. CD spectral changes and the sugar-induced pK(a) shift suggested that the microenvironment of the active site of these myoglobins was re-formed from a partially disturbed state to that comparable to the native state upon D-fructose binding. The correlation of pK(a) with k(cat) (for the aniline hydroxylase activity) and the ΔG(D)(H₂O)-k(cat) profile showed that these structural changes of Mb-(m-Bphe)₂ and Mb(PhBOH)₂ were closely related to their sugar-enhanced aniline hydroxylase activity. Thus, the results established that an incorporation of the artificial receptor molecule can be a valid methodology for the design of stimuli-responsive semiartificial enzymes.