title: TK1656, a thermostable l-asparaginase from Thermococcus kodakaraensis, exhibiting highest ever reported enzyme activity.
abstract: Two L-asparaginase homologs, TK1656 and TK2246, have been found in the genome of Thermococcus kodakaraensis. The gene encoding TK1656 consists of 984 nucleotides corresponding to a polypeptide of 328 amino acids. To examine the properties of TK1656, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. TK1656 exhibited high asparaginase activity (2350 U mg⁻¹) but no glutaminase activity. The enzyme also displayed the D-asparaginase activity but 50% to that of L-asparaginase. The highest activity was observed at 85°C and pH 9.5. TK1656 catalyzed the conversion of L-asparagine to L-aspartatic acid and ammonia following Michaelise-Menten kinetics with a K(m) and V(max) values of 5.5 mM and 3300 mmol min⁻¹ mg⁻¹, respectively. The activation energy from the linear Arrhenius plot was found to be 58 kJ mol⁻¹. Unfolding studies suggested that urea could not induce complete unfolding and inactivation of TK1656 even at a concentration 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity.
authors: School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan.
authors: Shahid Mahmood
authors: SM
authors: Chohan
authors: Naeem
authors: N
authors: Rashid
mesh_terms: D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D001215:Asparaginase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011489:Protein Denaturation / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D014508:Urea / Q000494:pharmacology
chemical_list: D001224:Aspartic Acid; D001216:Asparagine; D000641:Ammonia; D014508:Urea; D001215:Asparaginase; D019791:Guanidine
keywords: Structural stability; Thermal stability; Thermococcus kodakaraensis; Unfolding; l-Asparaginase