Srere 9418565 In order to study the problem of metabolite channelling, a recent and elegant technique employed has been to link the DNA for two enzymes and to express the fused protein in cells. We have used this latter technique to prepare a fusion protein of yeast mitochondrial citrate synthase (CS1) yeast mitochondrial malate dehydrogenase (MDH1). Yeast cells which lack mitochondrial enzyme activity of the Krebs TCA cycle cannot grow on acetate (acetate-phenotype). When either CS1 or MDH1 is deleted from yeast cells (CS1- or MDH1- cells) or when a double deletion is made (CS1-/MDH1- cells) the cells cannot grow on acetate. The fusion protein restored growth on acetate when placed in yeast cells which lacked CS1, MDH1, and CS1 and MDH1. In addition, kinetic studies on the purified fusion protein indicated channelling of the intermediate oxalacetate (OAA). We propose to extend these studies by changing the order of the enzymes; by testing linkers of different lengths and sequences; and by using heterologous enzymes of CS and MDH. Nonsequential fusion proteins of Krebs TCA cycle enzymes will be made also. We will investigate the in vitro channeling assay using different OAA trapping systems and isotopic methods. %%% When cells are broken, one finds apparently mainly free enzymes which given rise to the idea that the cell is essentially a "bag of enzymes". There are many indications, however, that in the cell, sequential metabolic enzymes are in complexes which dissociate upon cell rupture. We have studied the interactions of sequential enzymes and their consequences in a variety of ways, including biophysical and genetic techniques. This grant proposal is to continue our studies on a fused two sequential enzyme systems, yeast mitochondrial malate dehydrogenase (MDH), and yeast mitochondrial citrate synthase (CS). This fusion protein was isolated and its kinetic studied. The results indicated that channeling of the intermediate occurred. We propose to study the phenomenon by producing a series of other fusion proteins of MDH and CS with changed order, changed linkers, and heterologous MDH and CS. These studies will serve as models for possible metabolic advantages and regulatory properties of enzyme-enzyme interactions in cells. ***
