Na+/K+ exchange switches the catalytic apparatus of potassium-dependent plant L-asparaginase

Plant-type L-asparaginases, which are a subclass of the Ntn-hydrolase family, are divided into potassium-dependent and potassium-independent enzymes with different substrate preferences. While the potassium-independent enzymes have already been well characterized, there are no structural data for any of the members of the potassium-dependent group to illuminate the intriguing dependence of their catalytic mechanism on alkali-metal cations. Here, three crystal structures of a potassium-dependent plant-type l-asparaginase from Phaseolus vulgaris (PvAspG1) differing in the type of associated alkali metal ions (K+, Na+ or both) are presented and the structural consequences of the different ions are correlated with the enzyme activity. As in all plant-type l-asparaginases, immature PvAspG1 is a homodimer of two protein chains, which both undergo autocatalytic cleavage to alpha and beta subunits, thus creating the mature heterotetramer or dimer of heterodimers (alpha beta)(2). The alpha beta subunits of PvAspG1 are folded similarly to the potassium-independent enzymes, with a sandwich of two beta-sheets flanked on each side by a layer of helices. In addition to the 'sodium loop' (here referred to as the 'stabilization loop') known from potassium-independent plant-type asparaginases, the potassium-dependent PvAspG1 enzyme contains another alkali metal-binding loop (the 'activation loop') in subunit alpha (residues Val111-Ser118). The active site of PvAspG1 is located between these two metal-binding loops and in the immediate neighbourhood of three residues, His117, Arg224 and Glu250, acting as a catalytic switch, which is a novel feature that is identified in plant-type l-asparaginases for the first time. A comparison of the three PvAspG1 structures demonstrates how the metal ion bound in the activation loop influences its conformation, setting the catalytic switch to ON (when K+ is coordinated) or OFF (when Na+ is coordinated) to respectively allow or prevent anchoring of the reaction substrate/product in the active site. Moreover, it is proposed that Ser118, the last residue of the activation loop, is involved in the potassium-dependence mechanism. The PvAspG1 structures are discussed in comparison with those of potassium-independent l-asparaginases (LlA, EcAIII and hASNase3) and those of other Ntn-hydrolases (AGA and Tas1), as well as in the light of noncrystallographic studies.