L-asparaginase of Thermus thermophilus: purification, properties and identification of essential amino acids for its catalytic activity.
L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77 degrees C. Chemical modification experiments implied the existence ofhistidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.
Pritsa
A A
AA
Department of Chemistry, Aristotle University of Thessaloniki, Greece.
Kyriakidis
D A
DA
D000469:Alkaline Phosphatase / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D001215:Asparaginase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D011489:Protein Denaturation; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*; D013997:Time Factors
D000596:Amino Acids; D006639:Histidine; D001120:Arginine; D000469:Alkaline Phosphatase; D001215:Asparaginase