Enzymatically active truncated cat brain glutamate decarboxylase: expression, purification, and absorption spectrum

The DNA encoding the sequence for glutamate decarboxylase from cat brain was recloned into the Escherichia coli expression vector pET11a. The N-terminal 77- to 84-amino acid residues encoded by the cloned gene had been deleted from the protein which was purified to near homogeneity in 20-mg batches. The truncated protein is a dimer with a subunit molecular mass of about 59 kDa. This protein is enzymatically active and has a Km for L-glutamate of 1.37 mM and a turnover number of 7 s-1 at its optimal pH of 6.6. The absorption spectrum, resulting from the bound coenzyme, pyridoxal phosphate, showed pH-dependent bands at 338 and 420 nm with an isosbestic point at 356 nm. A spectrophotometric pKa value of 6.92 was evaluated for the bound coenzyme. The pH-dependent kinetic data suggest the presence of two dissociable groups in the free enzyme with pKa values of about 6.45 and 7.05 and pKa value of the enzyme-substrate complex of about 6.82 in phosphate buffer. Structures for the coenzyme in the active site of brain glutamate decarboxylase are proposed.