PREPARATION AND KINETIC CHARACTERIZATION OF A FUSION PROTEIN OF YEASTMITOCHONDRIAL CITRATE SYNTHASE AND MALATE-DEHYDROGENASE

We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA(-). The fusion protein (CS1/MDH1) is the C-terminus of CS1 l inked in-frame to the N-terminus of MDH1 with a short linker of glycyl -seryl-glycyl. The fusion protein produced was isolated and purified. Gel filtration studies indicated that CS1/MDH1 had a M(r) of similar t o 170 000. Western blotting analysis with SDS gel indicated a M(r) of similar to 90 000-95 000 (theoretical M(r) = 87 000). This is the expe cted M(r) for the fusion protein subunit. The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free e nzymes. In addition, the effect of AAT reaction, as a competitor for t he intermediate OAA of the coupled MDH-CS reaction, was examined. It w as observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are emp loyed. In addition, the transient time for the coupled reaction sequen ce was less for the fusion protein than for the free enzymes.