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 pKa 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 pKa 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 pKa with kcat (for the aniline hydroxylase activity) and the delta GDH2O-kcat 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.
Hamachi
I
I
Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka, Japan. itarutcm@mbox.nc.kyushu-u.ac.jp
Nagase
T
T
Tajiri
Y
Y
Shinkai
S
S
D000815:Aniline Hydroxylase / Q000138:chemical synthesis* / Q000378:metabolism*; D001897:Boronic Acids / Q000138:chemical synthesis* / Q000378:metabolism; D002241:Carbohydrates / Q000737:chemistry*; D004355:Drug Stability; D007700:Kinetics; D009211:Myoglobin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism; D010452:Peptide Biosynthesis; D011955:Receptors, Drug / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry
D001897:Boronic Acids; D002241:Carbohydrates; D009211:Myoglobin; D011955:Receptors, Drug; D014867:Water; D000815:Aniline Hydroxylase; C010686:benzeneboronic acid