title: Tailoring structure-function properties of L-asparaginase: engineering resistance to trypsin cleavage.
abstract: Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg206-Ser207 of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme.
authors.affiliation: Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855-Athens, Greece.
authors.forename: Georgia A
authors.initials: GA
authors.lastname: Kotzia
authors.forename: Katerina
authors.initials: K
authors.lastname: Lappa
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
mesh_terms: D001215:Asparaginase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D014357:Trypsin; D001215:Asparaginase