Related Experiment Videos
Lysine: N6-hydroxylase: stability and interaction with ligands
Journal of Protein Chemistry
|June 6, 2000
Summary
Recombinant lysine:N6-hydroxylase (rIucD) loses function via aggregation but retains structure. The C-terminal segment is essential for maintaining its native conformation and catalytic activity.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Recombinant lysine:N6-hydroxylase (rIucD) is an apoenzyme requiring FAD and NADPH for function.
- rIucD undergoes time-dependent aggregation and loss of monooxygenase activity, reversible by thiols.
Purpose of the Study:
- To investigate ligand-induced conformational changes in rIucD.
- To determine the role of the C-terminal segment in rIucD's structure and function.
Main Methods:
- Circular Dichroism (CD) spectroscopy
- Differential Scanning Calorimetry (DSC)
- Protease susceptibility assays (endo- and exopeptidases)
- Carboxypeptidase Y treatment
- Site-directed mutagenesis
Main Results:
- CD spectra and DSC profiles showed no significant conformational changes upon ligand binding.
- FAD and ADP protected rIucD from protease degradation, while NADPH offered partial protection and L-lysine was ineffective.
- Deletion of the C-terminal segment, via enzymatic treatment or mutagenesis, abolished rIucD's monooxygenase activity.
Conclusions:
- The C-terminal segment is critical for maintaining the native conformation of rIucD.
- While ligands like FAD do not induce major conformational shifts, they can protect the enzyme from degradation, highlighting the importance of the C-terminus for structural integrity and function.