Further studies on aspartate aminotransferase of thermophilic methanogens by analysis of general properties, bound cofactors, and subunit structures.
Aspartate aminotransferase (AspAT) [EC 2.6.1.1] of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M. thermoformicicum strain SF-4. AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M. thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate. The enzymes gave similar absorption spectra having maxima at around 326 and 415 nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit. Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor. Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes. Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form. Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4. They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.
Tanaka
T
T
Department of Biology, Faculty of Science, Osaka City University.
Yamamoto
S
S
Taniguchi
M
M
Hayashi
H
H
Kuramitsu
S
S
Kagamiyama
H
H
Oi
S
S
D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D017014:Methanobacterium / Q000201:enzymology*; D008970:Molecular Weight; D013045:Species Specificity; D013053:Spectrophotometry
D046911:Macromolecular Substances; D001219:Aspartate Aminotransferases