Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 862-868 |
| Number of pages | 7 |
| Journal | Bioconjugate Chemistry |
| Volume | 8 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - Nov 1 1997 |
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All Science Journal Classification (ASJC) codes
- Biotechnology
- Bioengineering
- Biomedical Engineering
- Pharmacology
- Pharmaceutical Science
- Organic Chemistry
Cite this
Incorporation of an artificial receptor into a native protein : New strategy for the design of semisynthetic enzymes with allosteric properties. / Hamachi, Itaru; Nagase, Tsuyoshi; Tajiri, Yusuke; Shinkai, Seiji.
In: Bioconjugate Chemistry, Vol. 8, No. 6, 01.11.1997, p. 862-868.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Incorporation of an artificial receptor into a native protein
T2 - New strategy for the design of semisynthetic enzymes with allosteric properties
AU - Hamachi, Itaru
AU - Nagase, Tsuyoshi
AU - Tajiri, Yusuke
AU - Shinkai, Seiji
PY - 1997/11/1
Y1 - 1997/11/1
N2 - 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.
AB - 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.
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U2 - 10.1021/bc970055z
DO - 10.1021/bc970055z
M3 - Article
C2 - 9404659
AN - SCOPUS:0031281620
VL - 8
SP - 862
EP - 868
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
SN - 1043-1802
IS - 6
ER -