9724922 Srere The investigator's laboratory has presented evidence in the past that the enzymes of the Krebs tricarboxylic acid cycle exist in vivo as a metabolon, i.e., a complex of sequential metabolic enzymes. Their most recent experimental approach is fusing malate dehydrogenase and citrate synthase. They have found that the best fusion protein for replacing the enzymes in a double mutant of yeast that lacks those two enzymes is one in which the C-terminus of mitochondrial citrate synthase is linked to the N-terminus of mitochondrial malate dehydrogenase. This protein was also produced and isolated using recombinant technology. The kinetics of the isolated fusion protein were studied and a channeling of oxalacetate was observed. They will extend these studies to include aconitase, the next enzyme in the metabolic sequence. The project has two specific aims. The first is to continue the kinetic studies on the fusion proteins of citrate synthase, aconitase, and malate dehydrogenase. The second concerns two forms of citrate synthase, the peroxisomal and mitochondrial, which have similar enzyme activities but different binding properties. As a preliminary step in comparing the ability of the peroxisomal form to substitute for the mitochondrial form in the mitochondria, they will investigate the reason it has not yet been possible to express peroxisomal citrate synthase activity in mitochondria. The methodologies to be used are those they have published previously on the citrate synthase-malate dehydrogenase fusion protein adapted to the inclusion of aconitase. The investigator's previous work provides evidence that in certain metabolic pathways, the component enzymes form a sequential complex which enhances their effciency. This phenomenon will be further explored in the current project. This enhancement of activity has many implications for basic biology and for the development of efficient biotechnological products.
