L-asparaginases (E.C.184.108.40.206, L- ASNases) have been widely used in clinical practice as chemotherapeutic drugs of acute lymphoblastic leukaemia (ALL). In order to evaluate the structural and functional role of selected residues in ASNases we report the screening of a library of L-ASNase mutants aiming to find detrimental mutations that significantly affect catalysis and substrate specificity. The library of mutants was created using the staggered extension process (StEp) and the genes of L- ASNases from Erwinia chrysanthemi (ErL-ASNase) and Erwinia carotovora (EcaL-ASNase). A mutant that displayed dramatic reduction in L-asparaginase activity and undetectable activity towards L-Gln and Nα - acetyl-L-Asn was isolated and characterized. Sequence of the mutant showed that it has a single point aminoacid replacement (Gly281Ser) of the E. carotovora enzyme. Steady-state kinetic analysis demonstrated that the Gly281Ser aminoacid replacement influence significantly the enzyme’s structural and functional properties. In particular it displays 10.8-fold increase in Km and 45.5-fold lower catalytic activity towards L-Asn. Analysis of the pH dependence of Vmax and Vmax/Km of L-Asn hydrolysis showed that the mutant displays modified properties regarding the dependence of kinetic constants towards the pH. Studies on the thermal stability of the mutant enzyme demonstrated that it exhibits about 3.8°C higher halfinactivation temperature, compared to the wild-type enzyme, suggesting that Gly281 contributes to the low stability of the enzyme. Biocomputing analysis suggested that the Gly281Ser replacement causes indirectly changes in the active site architecture and presumably alters the dynamics of the enzyme.
Keywords: L-asparaginase, hydrolase, enzyme engineering, leukemia, directed evolution, substrate specificity, enzyme flexibility.
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