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Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from
P Cerdan1, M Rekik, S Harayama
1Department of Medical Biochemistry, University Medical Center, Geneva, Switzerland.
European Journal of Biochemistry
|April 1, 1995
Summary
Investigating catechol 2,3-dioxygenases revealed that a single His250Gln substitution in NahH significantly impacts 3-methylcatechol oxidation efficiency and suicide inhibition. This study elucidates key amino acid roles in enzyme activity and substrate specificity.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Catechol 2,3-dioxygenases (C23Os) are crucial enzymes in aromatic compound degradation.
- The TOL plasmid's XylE and NAH7 plasmid's NahH enzymes exhibit distinct substrate specificities, particularly for 3-methylcatechol.
- Differences in catalytic efficiency (kcat) and suicide inhibition (kinact) between XylE and NahH for 3-methylcatechol were observed.
Purpose of the Study:
- To identify the specific amino acid residues responsible for the differing substrate specificities of XylE and NahH.
- To elucidate the mechanisms underlying the reduced catalytic efficiency and increased suicide inhibition of NahH towards 3-methylcatechol.
- To investigate the relationship between enzyme structure and function in catechol 2,3-dioxygenases.
Main Methods:
- Comparative analysis of substrate specificities between XylE and NahH.
- Construction and characterization of NahH-XylE hybrid proteins.
- Determination of kinetic parameters (kcat and kinact) for 3-methylcatechol oxidation and suicide inhibition.
Main Results:
- The XylE enzyme efficiently catalyzes the ring-cleavage of catechol, 3-methylcatechol, and 4-methylcatechol.
- NahH exhibits partial deficiency in oxidizing 3-methylcatechol, characterized by lower kcat and higher susceptibility to suicide inhibition.
- A single amino acid substitution (His250Gln in NahH) was identified as a primary determinant for reduced kcat and increased kinact.
- Substitutions at residues 77-102 in NahH also contribute to differences in suicide inhibition rates.
- The binding site for 3-methylcatechol during suicide inhibition differs from the catalytic site.
Conclusions:
- The His250Gln substitution is critical for the altered kinetic properties of NahH towards 3-methylcatechol.
- Enzyme structure, specifically amino acid residues in the N-terminal region, significantly influences substrate specificity and inhibition.
- Understanding these molecular determinants provides insights into enzyme evolution and engineering for biodegradation applications.