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B6db references: 94361514

type Journal Article
authors Chu, W. C.; Metzler, D. E.
title Enzymatically active truncated cat brain glutamate decarboxylase: expression, purification, and absorption spectrum
journal Arch Biochem Biophys
ui 94361514
year (1994)
volume 313
number 2
pages 287-95.
keywords Animal
abstract 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.
last changed 2002/11/12 16:17

B6db references