ABSTRACTpropose that they should be more correctly termed sucrolysis and sucroneogenesis. Before recent work it was customary to assume that sucrose synthase action resulted in the formation of UDP-glucose and then other nucleotide sugars leading into sugar polymer synthesis, such as plant cell walls. However, a substrate level pool of PPi was measured in plants (2,7,27), and we successfully tested the pyrophosphorolysis of UDPglucose feeding glucose 1-P directly into plant metabolism (32). These steps are an integral part of the recently proposed sucrose synthase pathway (1,15,30). Then, of course, from glycolysis carbon can be directed into essentially every metabolic activity of a cell. Therefore, the ability of a tissue or organ to metabolize sucrose must be one determinant of sink strength. Here we have tested the feasibility of biochemically measuring sucrose sink strength by assaying sucrose cleavage activities, i.e. sucrolysis via either the invertases or by the sucrose synthase pathway. The reasons we developed this UDP and PPi-dependent assay for sucrose synthase activity were (a) to measure activity in the sucrose breakdown direction whereas many other workers measured the opposite, namely sucrose synthesis (3,4,22,26), and (b) others who measured sucrose breakdown often assayed for UDP-glucose accumulation as a precursor of the synthesis of cell walls or other nucleotide sugars (6, 22). But in our assay we couple through the PPi-dependent UDP-glucopyrophosphorylase, which is very active in plants, to the formation of glucose 1-P which feeds carbon directly into glycolysis and possibly on to starch formation (31,32

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100°C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO 2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent K m values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90°C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80°C (pH 8.0), the apparent V m value for pyruvate decarboxylation was about 40% of the apparent V m value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.

Plant cells have two cytoplasmic pathways of glycolysis and gluconeogenesis for the reversible interconversion of fructose 6‐phosphate (F‐6‐P) and fructose 1,6‐bisphosphate (F‐1,6‐P2). One pathway is described as a maintenance pathway that is catalyzed by a nucleotide triphosphate‐dependent phosphofructokinase (EC 2.7.1.11; ATP‐PFK) glycolytically and a F‐1,6 bisphosphatase (EC 3.1.3.11) gluconeogenically. These are non‐equilibrium reactions that are energy consuming. The second pathway, described as an adaptive pathway, is catalyzed by a readily reversible pyrophosphate‐dependent phosphofructokinase (EC 2.7.1.90; PP‐PFK) in an equilibrium reaction that conserves energy through the utilization and the synthesis of pyrophosphate. A constitutive regulator cycle is also present for the synthesis and hydrolysis of fructose 2,6‐bisphosphate (F‐2,6‐P2) via a 2‐kinase and a 2‐phosphatase, respectively. The pathway catalyzed by the ATP‐PFK and F‐1,6‐bisphosphatase, the maintenance pathway, is fairly constant in maximum activity in various plant tissues and shows less regulation by F‐2,6‐P2. Plants use F‐2,6‐P2 initially to regulate the adaptive pathway at the reversible PPi‐PFK step. The adaptive pathway, catalyzed by PPi‐PFK, varies in maximum activity with a variety of phenomena such as plant development or changing biological and physical environments. Plants can change F‐2,6‐P2 levels rapidly, in less than 1 min when subjected to rapid environmental change, or change levels slowly over periods of hours and days as tissues develop. Both types of change enable plants to cope with the environmental and developmental changes that occur during their lifetimes. The two pathways of sugar metabolism can be efficiently linked by the cycling of uridylates and pyrophosphate required for sucrose breakdown via a proposed sucrose synthase pathway. The breakdown of sucrose via the sucrose synthase pathway requires half the net energy of breakdown via the invertase pathway. Pyrophosphate occurs in plant tissues as a substrate pool for biosynthetic reactions such as the PPi‐PFK or uridine diphosphate glucose pyrophosphorylase (EC 2.7.7.9; UDPG pyrophosphorylase) that function in the breakdown of imported sucrose. Also, pyrophosphate links the two glycolytic/gluco‐neogenic pathways; and in a reciprocal manner pyrophosphate is produced as an energy source during gluconeogenic carbon flow from F‐1,6‐P2 toward sucrose synthesis.

Here it is reported that sucrose synthase can be readily measured

1988. A reassessment of glycolysis and gluconeogenesis in higher plants. -Physiol. Plant. 72: 650-654.Sucrose is the starting point of glycolysis and end point of gluconeogenesis in higher plants. During both glycolysis and gluconeogenesis alternative enzymes are present at various steps to carry out parallel pathways; alternatives are available for utilizing nucleotide triphosphates and pyrophosphate; fructose 2.6-bisphosphate serves as a strong internal regulator; and plants use these cytoplasmic alternatives as they develop and as their environments change.