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 engin eered myoglobins bearing a phenylboronic acid moiety, which were semis ynthesized by a cofactor reconstitution method, were studied by the de naturation experiment, spectrophotometric titration of the pK(a) shift of the a,dal H2O, circular dichloism (CD), and the kinetics of the my oglobin-catalyzed-aniline hydroxylation reaction. Both boronophenylala nine-appended myoglobin [Mb(m-Bphe)(2)] and phenylboronic acid-appende d 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 stat e 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 Delta G(D)(H2O)-k(cat) profile showed that these structural changes of Mb(m-Bphe)(2) and Mb(PhBOH)(2) were closely related to thei r sugar-enhanced aniline hydroxylase activity. Thus, the results estab lished that an incorporation of the artificial receptor molecule can b e a valid methodology for the design of stimuli-responsive semiartific ial enzymes.