Interaction between Muscle Aldolase and Muscle Fructose 1,6-Bisphosphatase Results in the Substrate Channeling

Abstract

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.

Colocalization of aldolase and FBPase in cytoplasm and nucleus of cardiomyocytes

“…On the other hand, enzymes catalyzing reversible reactions, like aldolase, might create either glycolytic or glyconeogenic complexes, depending on cellular requirements (Rakus et al, 2004;Mamczur et al, 2005). In the glycogen synthesis, aldolase not only provides the substrate for FBPase but also protects it against AMP inhibition (Rakus and Dzugaj, 2000;Rakus et al, 2003b).…”

Enzyme co-localization in pea leaf chloroplasts: glyceraldehyde-3-P dehydrogenase, triose-P isomerase, aldolase and sedoheptulose bisphosphatase

“…There is also evidence for physical interaction between these enzymes in animal systems (Pontremoli et al 1979;MacGregor et al 1980;Rakus et al 2003;Dziewulska-Szwajkowska et al 2004). Addition of a mutant fructose bisphosphatase without enzyme activity to a system composed of rabbit muscle aldolase and fructose bisphosphatase results in a reduction in the steady state rate of fructose-6-P formation, indicating that fructose bisphosphate can, in fact, be channeled between the two muscle enzymes in vitro (Rakus et al 2004). There are three types of Class I aldolase in vertebrates, type A, characteristic of muscle cells and primarily involved in glycolysis, type B, characteristic of liver and primarily involved in gluconeogenesis, and the C type, characteristic of brain tissue.…”

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

“…Passage of an intermediate directly from one active site to another without equilibration with the solvent is called channeling (26)(27)(28)(29)(30). Examples of reactive intermediates that are channeled from one active site to another include carbamate (31), ammonia (32), indole (33), aspartyl phosphate (34), glutamyl phosphate (35), and phosphoribosylamine (26).…”