9407122 Kohl The oxidative limb of the pentose phosphate pathway (PPP) in the cytoplasm of soybean nodules may be an important source of ribose- 5-phosphate, the starting point for the synthesis of ureades and the form in which biologically fixed nitrogen is exported from the nodule to the whole plant. Results with soybean suggesting that glucose-6-phosphate (G6P) in nodule extract is not in equilibrium with the concentration of G6P in bulk medium, and that half of the PPP activity precipitates at a relatively modest centrifugal force (290,000 x g) have led to the proposition that the PPP functions as a macromolecular complex (metabolon). Within this metabolon, PPP intermediates are channeled from one enzyme to the next. Examples of metabolic channeling in other systems has proved to be important in conserving the limited solvent capacity of cell water, protecting intermediates which are unstable if not enzyme bound, and maintaining metabolite concentration gradients inside cells. If our hypothesis is correct about PPP channeling, this example of "channeling" would contribute not only to understanding about nodule metabolism but also to understanding pentose phosphate metabolism in other plant and animal cells. Several approaches will be used to investigate the idea of PPP channeling including (i) determining which PPP intermediates are out of equilibrium with their counterparts in the bulk medium, (ii) measuring "metabolon" size via its centrifugal and gel filtration behavior, and (iii) comparing the kinetics of pathway function by metabolons vs. "collections" of dissociated enzymes. %%% Metabolic pathways consist of a series of reactions, each catalyzed by a specific enzyme. The product of one reaction becomes the substrate (food) of the next enzyme until a final product is formed. In some cases, the enzymes of a pathway are associated with each other such that the product of one reaction does not leave the site of "formation." In this way product(s) m ay be "channeled" directly from one enzyme to another. When this happens, channeling can be important in improving metaboliic efficiency by speeding up a series of reactions and protecting certain unstable intermediates on the way to a final product. Work in this laboratory has led to the hypothesis that enzymes of the pentose phosphate pathway, a key pathway in carbohydrate formation, operates as a macromolecular complex to channel intermediates from one enzyme to the next. The present work will test this idea and add to the body of evidence that similar enzyme arrangements occur in the metabolic pathways of other cells as well. ***
