Electrostatic Channeling of Oxaloacetate in a Fusion Protein of Porcine Citrate Synthase and Porcine Mitochondrial Malate Dehydrogenase

Abstract

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.

Diffusion of Tricarboxylic Acid Cycle Enzymes in the Mitochondrial Matrix in Vivo

“…The restricted rotational mobility of an enzyme (27) can be interpreted in terms of enzyme binding to the mitochondrial inner membrane rather than complexation with other enzymes. The increased metabolic rate after genetic fusion of successive enzymes in a pathway (13)(14)(15) is not surprising and does not prove that complexation occurs in vivo. Finally, the rapid diffusion of unconjugated GFP in the mitochondrial matrix (29) suggests that metabolite channeling may not be required to overcome diffusive barriers if metabolites, like the much larger GFP, are able to diffuse rapidly in the matrix.…”

“…10 -12). Genetic fusions of the sequential tricarboxylic acid cycle enzymes malate dehydrogenase and citrate synthase from yeast (13,14) and pig (15) gave improved kinetics compared with similar solutions of the unconjugated enzymes. Numerical simulations provided evidence for a continuous surface channel that may facilitate electrostatic channeling of substrate between the active sites of three consecutive tricarboxylic acid cycle enzymes (malate dehydrogenase, citrate synthase, and aconitase) (16,17).…”

Voluntary resistance running with short distance enhances spatial memory related to hippocampal BDNF signaling

“…An increase in citrate synthase (CS) activity, which reflects an improved aerobic metabolism in mitochondria of skeletal muscle, is commonly used to confirm exercise-training effects. Therefore, the CS activity in the plantaris and soleus muscles was measured using a method described previously (41). The CS activity in muscles was determined in duplicate using the CS Assay Kit (Sigma, St. Louis, MS) following the manufacturer's instructions.…”

Reversible Transdominant Inhibition of a Metabolic Pathway

“…3). In the last few years, evidence was presented concerning the channeling of oxaloaxetate in vitro between yeast mitochondrial CS (Cit1p) and MDH (Mdh1p) (6) as well as between the corresponding pig enzymes (7,8).…”