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Rat liver phenylalanine hydroxylase. Activation by sulfhydryl modification
The Journal of Biological Chemistry
|July 10, 1981
Summary
Modifying phenylalanine hydroxylase with N-ethylmaleimide significantly boosts its activity and alters its kinetic properties. This chemical modification mimics natural activation states, offering insights into enzyme regulation.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein modification
Background:
- Phenylalanine hydroxylase (PAH) is a key enzyme in phenylalanine metabolism.
- Understanding PAH regulation is crucial for metabolic disorders like phenylketonuria.
- Previous studies indicated activation by proteolysis or phospholipids.
Purpose of the Study:
- To investigate the effect of N-ethylmaleimide (NEM) modification on PAH activity and kinetics.
- To compare NEM-modified PAH characteristics with other known activated forms of the enzyme.
Main Methods:
- Chemical modification of phenylalanine hydroxylase using N-ethylmaleimide.
- Assay of hydroxylase activity using tetrahydrobiopterin as a cofactor.
- Kinetic analysis of phenylalanine binding and substrate specificity.
- Comparison of native and modified enzyme behavior.
Main Results:
- N-ethylmaleimide selectively modifies a single sulfhydryl residue on the PAH subunit.
- This modification results in a 20-30 fold increase in hydroxylase activity.
- NEM-modified PAH exhibits hyperbolic kinetics, broadened substrate specificity, and dependence on stimulator protein, similar to proteolytically or phospholipid-activated PAH.
- Phenylalanine binding shifts from sigmoidal to hyperbolic with decreased overall binding capacity.
Conclusions:
- N-ethylmaleimide modification serves as a chemical tool to activate phenylalanine hydroxylase.
- The observed changes in kinetics and binding suggest a conformational change induced by sulfhydryl modification.
- This study provides further evidence for the complex regulatory mechanisms of phenylalanine hydroxylase.
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