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Histidine 282 in 5-aminolevulinate synthase affects substrate binding and catalysis
Tracy D Turbeville1, Junshun Zhang, Gregory A Hunter
1Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612, USA.
Biochemistry
|May 2, 2007
Summary
Histidine 282 in 5-aminolevulinate synthase (ALAS) is crucial for heme biosynthesis. Its mutation significantly impairs enzyme function and cofactor interaction, impacting the PLP-dependent pathway.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- 5-Aminolevulinate synthase (ALAS) is a key enzyme in heme biosynthesis.
- It belongs to the pyridoxal 5'-phosphate (PLP)-dependent alpha-oxoamine synthase family.
- A conserved histidine residue is implicated in cofactor binding and enzyme activity.
Purpose of the Study:
- To investigate the role of histidine 282 (H282) in ALAS function.
- To elucidate the impact of H282 mutation on PLP cofactor interaction and catalytic efficiency.
Main Methods:
- Site-directed mutagenesis (H282A substitution).
- Enzyme kinetics assays (measuring catalytic efficiency and binding constants).
- Spectroscopic analyses (UV-Vis absorption, fluorescence, circular dichroism).
Main Results:
- H282A mutation reduced catalytic efficiency for glycine 450-fold.
- Glycine binding rate decreased by 85%, and PLP binding affinity increased 9-fold.
- Spectroscopic changes indicated altered tautomer distribution and impeded PLP reorientation upon substrate binding.
Conclusions:
- H282 is essential for ALAS activity, coordinating PLP movement and active site reorganization.
- It acts as a hydrogen bond donor, maintaining the Schiff base and enhancing PLP's electron sink function.
- These findings clarify the catalytic mechanism of PLP-dependent enzymes in heme biosynthesis.
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