[Transaminase B from Escherichia coli. I.-Purification and first properties].
Transaminase B (EC.2.6.1.6.) from E. coli, the specific enzyme for branched-chain aminoacids, was obtained in a purity equal to or greater than 96 p. cent after an 800-fold purification, employing two different procedures. One of the procedures involved heating at 60degreesC. The apparent molecular weight of the enzyme was estimated by chromatography on Sephadex and gel electrophoresis to be close to 180,000. The protein is made up of 6 subunits of equal size, with one molecule of coenzyme in each. Its absorption spectrum shows bands at 335 and 415 nm, and was found to be almost insensitive to the pH of the medium between 4.6 and 9. Transaminase B is active on phenylalanine as well, although the reaction between L-phenylalanine and alpha-ketoglutarate is about 50 to 100 times slower than the analogous reaction using L-valine as an aminoacid. Three sets of data show that the phenylalanine aminotransferase activity associated with transaminase B is not an artefact due to a protein contaminant. 1) Activities displayed toward phenylalanine and valine cannot be resolved by different methods, including chromatography, gel electrophoresis, and electrofucussing. 2) The absorption spectrum of the enzyme is as strongly modified by phenylalanine as by valine. 3) A ketoglutarate-free reaction between phenylalanine, tyrosine or typtophane and an aliphatic alpha-ketoacid is catalyzed by the pure enzyme and follows a mechanism belonging to the usual ping pong type. The possible significance of this reaction as a regulatory device in the cell metabolism is briefly discussed.
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D008970:Molecular Weight; D010649:Phenylalanine / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D000637:Transaminases / Q000302:isolation & purification*; D014633:Valine / Q000378:metabolism
D010649:Phenylalanine; D000637:Transaminases; D014633:Valine
Transaminase B d'Escherichia coli. I. - Purification et premières propriétés