Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.
Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.
Sung
M H
MH
Laboratory of Microbial Biochemistry, Kyoto University, Japan.
Tanizawa
K
K
Tanaka
H
H
Kuramitsu
S
S
Kagamiyama
H
H
Soda
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001219:Aspartate Aminotransferases / Q000302:isolation & purification* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006358:Hot Temperature; D006882:Hydroxyapatites; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D000596:Amino Acids; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D017886:Durapatite; D001219:Aspartate Aminotransferases