A serine residue is responsible for catalytic activation of potassium-dependent plant asparaginase.

L-asparaginases play an important role in nitrogen mobilization in plants. Here, we investigated the biochemical and biophysical properties of potassium dependent (PvAspG1) and potassium independent (PvAspG-T2) L-asparaginases from Phaseolus vulgaris. Our previous studies revealed that PvAspG1 requires potassium for catalytic activation and its crystal structure suggested that Ser-118 in the activation loop plays a critical role in coordinating the metal cation. This amino acid residue is replaced by an isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, secondary structure conformation, allosteric effector binding and pH profile were studied. Introduction of the serine residue conferred potassium activability to PvAspG-T2. Conversely, the PvAspG1-S118I mutant was no longer activable by potassium. PvAspG1 and PvAspG-T2-I117S had a similar apparent Km value for K+, of 0.2-0.4 mM. The mutations did not result in structural instability, as determined by circular dichroism spectroscopy of thermal unfolding. Potassium elicited a similar conformational change in PvAspG1 and PvAspG-T2-I117S, as determined by circular dichroism spectroscopy. However, no change in conformation was observed for PvAspG-T2 and PvAspG1-S118I. The presence of the serine residue was associated with a narrow pH profile, with a sharp peak in enzyme activity between pH 6.5-7 and pH 8.0, suggesting that potassium influences the ionization of catalytic residues at the active site. Together, these results indicate that Ser-118 of PvAspG1 is essential and sufficient for potassium activation in plant L-asparaginases.