The enzymes of the Krebs TCA cycle may form a multienzyme complex, creating channels through which substrates and intermediates may be transported between enzymes. To test this model, we constructed two fusion proteins containing yeast mitochondrial CS (CS1) and MDH (MDH1) moieties. In both fusion proteins the C terminus of CS1 is connected to the Ntenninus of MDH 1 either by a linker of 3 or 10 a.a. These fusion proteins show channeling activity in competition reactions not shown by a mixture of free CS1 and MDH1. These results confirm a recent model of electrostatic interactions of these enzymes (Elcock tk McCammon, Biochem, 1996)and an earlier prediction trom this laboratory (Lindbladh rl at. Bincheni 1994). The model predicts the formation of an electropositive channel for transport oi'the intermediate oxalacetate from the active site of one enzyme to the active site oi another. To test this hypothesis we studied the influence oi ionic strength on the function of fusion proteins in a < ompetition reaction. An increase in ionic strength results in the decrease of channeling activity of fusion proteins in competition reactions to the level of activity of a mixture of free enzymes. Thus, high ionic strength caused the disruption of the channel. These observations support the model of formation of an electrostatic channel which can transport intermediate substrates from one enzyme active site to another.
|Publication status||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology