Role of asparaginase variable loop at the carboxyl terminal of the alpha subunit in the determination of substrate preference in plants

Abstract

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, Phe¹⁶², preceding the variable loop, whose side chain is located in proximity to the beta-carboxylate group of the product aspartate, and to Gly²⁴⁶, 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.