*3.6. Cytosolic Pyruvate Kinases are Regulated by Subgroup Association and Dissociation*

Most glycolytic regulatory enzymes, including PFK, PEPC and PK, were shown to exist as oligomers, whereby many of them can reversibly dissociate after binding effector molecules. This is often accompanied by an altered enzyme activity and therefore provides a mechanism for regulation [33]. Our catalytic characterization of cPK enzymes supports this hypothesis, since single isoform activities are significantly lower than activities of equimolar mixtures of specific isoforms (Figure 7). For plastidial PK enzymes, it was even reported that single enzymes or their respective homo oligomers, have no activity at all [5]. Thus, enzyme oligomerization seems to be a common mechanism to regulated PK enzyme activity.

The determined enzyme pairs are based not simply on sequence homology, but they are more in agreement with the tissue-specific expression pattern of the respective isoforms observed in our GUS studies. Accordingly, the most pronounced effect on enzyme activity was observed when the isoforms cPK4 and cPK5 were combined (Figure 7), which both appear to be strongly induced by cold treatment. The observation that cPK2 and cPK3 have positive impact on the activity of each other is underlined by co-expression data, which show that expression of *cPK2* and *cPK3* is strongly correlated (ATTEDII, version 7.1), indicating both isoforms to be active in the same metabolic pathway simultaneously. Interestingly, cPK1 appears to take over a special regulatory role because it exhibited the lowest specific activity as single enzyme (Table 1), but it was able to increase the activity of other isoforms in different combinations (Figure 7). Furthermore, cPK1 seems to underlie the most multisided metabolic control (Table 2) and appears to be most generally expressed isoform, independent of tissue and developmental stages. Finally, this supports the hypothesis of a further regulatory aspect, as *cPK1* expression might be sufficient to meet energy demand alone under certain conditions, whereas a second isoform is expressed additionally at raised energy need.
