*2.4. Pyruvate Kinase Expression Follows a Diurnal Course*

Carbon metabolism is exposed to diurnal changes, since photosynthesis cannot maintain energy provision during the dark period. Breakdown of starch and soluble sugars during the night leads to increased activity of glycolytic enzymes. In order to assess cPK activity, which is a key enzyme in glycolysis, in the course of the day/night cycle, *cPK* promoter GUS plants were sampled at the end of the night (after 8 h darkness) and at the end of the day (after 16 h light). Apart from differences in signal intensity between the lines, *cPK1*, *cPK2* and *cPK3* promoters lead to increased GUS activity when sampled after 8 h of darkness compared to plants sampled after 16 h in the light (Figure 6). In contrast, *cPK4* and *cPK5* promoter-GUS lines showed no detectable GUS activity in mature plants used for the day/night cycle experiment (data not shown).

**Figure 6.** Histochemical GUS expression in dependence on the daytime in 4-week-old plants expressing the *cPK1*-promoter-GUS, *cPK2*-promoter-GUS and *cPK3*-promoter-GUS constructs, grown on soil sampled after 16 h light and after 8 h darkness. Representative images of one out of three different transgenic lines are shown.

#### *2.5. Kinetic Characterization of Cytosolic Pyruvate Kinases*

To assess the kinetic parameters of cytosolic PK enzymes, coding sequences were cloned into the pET16B expression vector mediating N-terminal 6x-His tag. PK isoenzymes were expressed heterologously in *E. coli* and purified in order to obtain a sufficient yield (Appendix A Figure A4). Enriched isoenzymes were characterized in vitro applying a lactate dehydrogenase coupled enzyme assay according to [18]. Plots showing cPK activity versus linear dilution series of ADP and PEP describe hyperbolic saturation curves; thus, biochemical properties like Michaelis constant *Km*, enzymes maximum rate *Vmax*, and turnover number *kcat* were calculated applying the Michaelis–Menten equation (Table 1; Appendix A Figure A5). The lowest specific activity was observed for cPK1 with a *Vmax* of 1.4U/mg. On the contrary, PK5 exhibited the highest specific activity (*Vmax*: 6.4 U/mg). The *Km* values for ADP were in about the same range varying between 0.07 mM (cPK4) and 0.34 mM (cPK5). cPK2 had a quite high *Km* for PEP (0.76 mM), compared to the other isoenzymes.


**Table 1.** In vitro catalytic properties of cPK enzymes heterologously expressed in *Escherichia coli*: Michaelis constant *Km*, enzymes maximum rate *Vmax* and turnover number *kcat*.

#### *2.6. Allosteric E*ff*ects on Pyruvate Kinase Enzyme Activity*

PK enzymes are tightly regulated by allosteric effectors as known from other organisms [6,8,18]. To assess putative regulatory impact of diverse selected metabolites on cytosolic PK enzymes from *Arabidopsis thaliana*, single enzyme extracts were tested under substrate saturating conditions. ATP

showed a strong effect on cPK activity. In the case of cPK1, the specific activity was reduced by more than 90%, and for cPK3 a reduction of 73% was observed (Table 2). In contrast, ATP did not affect the specific activity of cPK5. Our data indicate that cPK1 can be positively affected by FBP, whereby glutamate and aspartate lead to a decrease in specific enzyme activity. Activities of other cPK isoenzymes were not significantly altered by the selected metabolites. Citrate is a central metabolite of the citrate cycle and has been applied as negative control in several previous kinetic analyses of pyruvate kinases [10]. Also, in our study citrate had a strong negative allosteric effect on all five isoforms.

**Table 2.** Allosteric effects on cPK enzyme activity after application of F1.6BP (1 mM), serine (0.2 mM), AMP (0.1 mM), ATP (2 mM), glutamate (0.2 mM), aspartate (0.2 mM) and citrate (4 mM) compared to control.

