Structural determinants responsible for the substrate preference of the potassium-independent (ASPGA1) and -dependent (ASPGB1) asparaginases from Arabidopsis thaliana have been investigated. Like ASPGA1, ASPGB1 was found to be catalytically active with both L: -Asn and β-Asp-His as substrates, contrary to a previous report. However, ASPGB1 had a 45-fold higher specific activity with Asn as substrate than ASPGA1. A divergent sequence between the two enzymes forms a variable loop at the C-terminal of the alpha subunit. The results of dynamic simulations have previously implicated a movement of the C-terminus in the allosteric transduction of K(+)-binding at the surface of LjNSE1 asparaginase. In the crystal structure of Lupinus luteus asparaginase, most residues in this segment cannot be visualized due to a weak electron density. Exchanging the variable loop in ASPGA1 with that from ASPGB1 increased the affinity for Asn, with a 320-fold reduction in K (m) value. Homology modeling identified a residue specific to ASPGB1, Phe(162), preceding the variable loop, whose side chain is located in proximity to the beta-carboxylate group of the product aspartate, and to Gly(246), a residue participating in an oxyanion hole which stabilizes a negative charge forming on the side chain oxygen of asparagine during catalysis. Replacement with the corresponding leucine from ASPGA1 specifically lowered the V (max) value with Asn as substrate by 8.4-fold.