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.
Chu
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WC
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Metzler
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DE
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002415:Cats; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D005822:Genetic Vectors; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013552:Swine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase