Diurnal accumulation of K+-dependent L-asparaginase in leaf of common bean (Phaseolus vulgaris L.).

L-Asparaginase (EC 3.5.1.1) activity has been previously reported to fluctuate with the photoperiod in young pea leaves, with higher activity in the light. The present research sought to investigate this phenomenon in developing leaves of common bean (Phaseolus vulgaris L.). There are two genes coding for K+-dependent asparaginase in this species. Expression of PvASPG1 predominates over PvASPG2 in all tissues. The catalytic efficiency of recombinant PvASPG2 was approximately 2-fold lower than that of PvASPG1. Polyclonal antibodies were raised against a specific peptide present in PvASPG1 to use in immunoblotting. In developing seed, asparaginase protein levels in the seed coat stayed constant, whereas levels in cotyledon were lower and progressively declined. In young leaf, asparagine protein levels showed diurnal variation, increasing at the end of the dark period and slowly decreasing during the light period. This was paralleled by changes in activity levels in leaf extracts. These changes accompanied a transient increase in free asparagine concentration at the beginning of the light period. The present results demonstrated that K+-dependent asparaginase activity reaches a maximum level at the transition from dark to light, anticipating dawn, in young leaves of common bean.

Structural basis of potassium activation in plant asparaginases.

l-asparaginases (EC 3.5.1.1) 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 isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, allosteric effector binding, secondary structure conformation, and pH profile were studied. Introduction of the serine residue conferred potassium activation in PvAspG-T2. Conversely, the PvAspG1-S118I mutant could no longer be activated by potassium. PvAspG1 and the PvAspG-T2-I117S mutant had a similar half-maximal effective concentration (EC50 ) value for potassium activation, between 0.1 and 0.3 mm. Potassium binding elicited a similar conformational change in PvAspG1 and PvAspG-T2-I117S, as studied by circular dichroism. However, no change in conformation was observed for PvAspG-T2 and PvAspG1-S118I. Analysis of kinetic parameters in function of pH indicated that potassium activation mediated by Ser-118 influences the ionization of specific functional groups in the enzyme-substrate complex. Together, the results indicate that Ser-118 of PvAspG1 is essential and sufficient for potassium activation in plant l-asparaginases. ENZYME: l-Asparaginase (EC 3.5.1.1).