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Enzymatically active truncated cat brain glutamate decarboxylase: expression, purification, and absorption spectrum
1Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Archives of Biochemistry and Biophysics
|September 1, 1994
Summary
Researchers purified an active, truncated glutamate decarboxylase enzyme from cat brain. This enzyme, crucial for neurotransmitter synthesis, exhibits specific kinetic properties and cofactor interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Glutamate decarboxylase (GAD) is a key enzyme in GABA synthesis.
- Understanding GAD's structure-function relationship is vital for neurological research.
Purpose of the Study:
- To express and purify a truncated form of cat brain glutamate decarboxylase.
- To characterize the enzymatic activity, kinetic parameters, and cofactor binding of the purified enzyme.
Main Methods:
- Recloning of cat brain GAD DNA into an E. coli expression vector.
- Purification of the truncated GAD protein.
- Enzymatic assays, spectrophotometry, and kinetic analysis.
Main Results:
- Purified truncated GAD dimer (59 kDa subunits) with significant enzymatic activity.
- Determined kinetic parameters: Km (1.37 mM), turnover number (7 s-1) at optimal pH (6.6).
- Spectroscopic analysis revealed pH-dependent coenzyme (pyridoxal phosphate) binding and pKa values.
Conclusions:
- The truncated GAD is enzymatically active and suitable for structural and functional studies.
- Kinetic data suggest specific amino acid residues are critical for catalysis and substrate binding.
- Proposed models for coenzyme binding within the active site provide insights into GAD mechanism.
