Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase.
Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bülow, L., Evans, C., and Srere, P.A (1994) Biochemistry 33, 11692-11698] that a fusion protein of yeast mitochondrial citrate synthase and yeast mitochondrial malate dehydrogenase channels oxaloacetate between the active sites. A Brownian dynamics simulation model of porcine mitochondrial enzymes of citrate synthase and malate dehydrogenase was used [Elcock, A. H., and McCammon, A. M. (1996) Biochemistry 35, 12652-12658], showing that a positive electrostatic surface potential between the active sites of the fusion protein could account for the channeling of oxaloacetate we observed with the yeast fusion protein. Since the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine fusion protein. The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein. This channeling behavior is eliminated at high ionic strength. A fusion protein of porcine citrate synthase and porcine cytosolic malate dehydrogenase does not exhibit any channeling of oxaloacetate. A model of the fusion protein with the cytosolic malate dehydrogenase shows no clear positive electrostatic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase. These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.
Shatalin
K
K
Pre-Clinical Science Unit, Dallas VA Medical Center, University of Texas Southwestern Medical Center at Dallas 75216, USA.
Lebreton
S
S
Rault-Leonardon
M
M
Vélot
C
C
Srere
P A
PA
D000818:Animals; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008958:Models, Molecular; D009994:Osmolar Concentration; D062907:Oxaloacetic Acid / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D055672:Static Electricity; D013552:Swine
D011993:Recombinant Fusion Proteins; D062907:Oxaloacetic Acid; D008291:Malate Dehydrogenase; D002950:Citrate (si)-Synthase