Cysteine synthase of an extremely thermophilic bacterium, Thermus thermophilus HB8.
O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol. The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein. The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.
Mizuno
Yasuko
Y
United Graduate School of Agricultural Sciences, Faculty of Agriculture, Gifu University, Japan.
Miyashita
Yoko
Y
Yamagata
Shuzo
S
Iwama
Tomonori
T
Akamatsu
Tsuyoshi
T
D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003547:Cysteine Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013440:Sulfides / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology*
D000596:Amino Acids; D001426:Bacterial Proteins; D007202:Indicators and Reagents; D013439:Sulfhydryl Reagents; D013440:Sulfides; D011732:Pyridoxal Phosphate; D003547:Cysteine Synthase