Fructose Inactive

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

In liver, cAMP activates gluconeogenesis, but in muscle, it activates glycolysis. Let s do liver first, and the muscle answer will just be the opposite. So, we want to activate gluconeogenesis in liver in response to increased phosphorylation (increased levels of cAMP). Phosphorylation of our enzyme (PFK-2) must have an effect that is consistent with the activation of gluconeogenesis. If gluconeogenesis is on and glycolysis is off, the level of fructose 2,6-bisphosphate (an activator of glycolysis) must fall. If fructose 2,6-bisphosphate is to fall, the PFK-2 that synthesizes it must be made inactive. So, in liver, phosphorylation of PFK-2 must inactivate the enzyme. 

Each 5 mL oral solution contains prednisolone 5 mg and alcohol 5% or 30%. Inactive ingredients include alcohol, citric acid, disodium edetate, fructose, hydrochloric acid, maltol, peppermint oil, polysorbate 80, propylene glycol,... 

Kaletra oral solution is available for oral administration as 80 mg lopinavir and 20 mg ritonavir per milliliter with the following inactive ingredients acesulfame potassium, alcohol, artificial cotton candy flavor, citric acid, glycerin, high fructose com symp, Magnasweet-110 flavor, men-... 

When fructose 2,6-bisphosphate binds to its allosteric site on PFK-1, it increases that enzyme s affinity for its substrate, fructose 6-phosphate, and reduces its affinity for the allosteric inhibitors ATP and citrate. At the physiological concentrations of its substrates ATP and fructose 6-phosphate and of its other positive and negative effectors (ATP, AMP, citrate), PFK-1 is virtually inactive in the absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate activates PFK-1 and stimulates glycolysis in liver and, at the same time, inhibits FBPase-1, thereby slowing gluconeogenesis. 

Dephosphorylated PFK-2 is active, whereas FBP-2 is inactive this favors formation of fructose 2,6-bisphosphate. 

Many enzymes are regulated by covalent modification, most frequently by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. In the fed state, most of the enzymes regulated by covalent modification are in Ihe dephosphorylated form and are active (see Figure 24.2). Three exceptions are glycogen phosphorylase (see p. 129), fructose bis-phosphate phosphatase-2 (see p. 98), and hormone-sensitive lipase of adipose tissue (see p. 187), which are inactive in their dephosphorylated state. 

The presence in mammalian liver of a specific phosphatase which catalyzes the hydrolysis of fructose 1,6-diphosphate (1) was first reported by Gomori in 1943 ( ). He succeeded in separating the enzyme from other phosphatases present in mammalian tissues and thus clarified much of the confusion which had previously existed regarding the specificity of these phosphatases. The specific fructosediphosphatase (FDPase) was shown to require a divalent cation such as Mg2+ and to be inactive at acid or neutral pH. It was present in the livers and kidneys of a number of mammalian species. 

The enzyme was purified from Candida utilis in 1965 by Rosen et al. (8Q). Dried yeast was allowed to autolyze in phosphate buffer at pH 7.5 for 48 hr, and the enzyme was isolated in crystalline form from these autolysates by a procedure which included heating to 55° at pH 5.0, fractionation with ammonium sulfate, and purification on phospho-cellulose columns from which the enzyme was specifically eluted with malonate buffer containing 2.0 mM FDP. Crystallization was carried out by addition of ammonium sulfate in the presence of mM magnesium chloride. The Candida enzyme was more active than the mammalian FDPases at room temperature and pH 9.5 the crystalline protein catalyzed the hydrolysis of 83 /nnoles of FDP per minute per milligram of protein. The enzyme was completely inactive with other phosphate esters, including sedoheptulose diphosphate, ribulose diphosphate, and fructose 1- or fructose 6-phosphates. Nor was the activity of the enzyme inhibited by any of these compounds. Optimum activity was observed at concentrations of FDP between 0.05 and 0.5 mM higher concentrations of FDP (5 mM) were inhibitory. 

A highly purified FDPase from the slime mold Polysphondylium pallidum has been shown (92), to hydrolyze both FDP and SDP, at nearly equal rates, to yield fructose 6-phosphate and sedoheptulose 7-phosphate, respectively. In other respects the purified enzyme was remarkably similar to that isolated from Candida utilis it was completely inactive at pH 7.5 or 8.0, and showed a pH optimum at 9.2. In the presence of low concentrations of EDTA a second pH optimum appeared at pH 7.5. Unlike the Candida FDPase, however, the Polysphondylium enzyme was not inhibited by AMP at any pH. The levels of enzyme which could be extracted from the cells did not change significantly during the various stages of differentiation, and its activity could not be related to catabolic or anabolic processes which characterize these stages. 

Where A is the initial product glucose, F the final product glyceraldehyde 3P, D1 and D3 the active and inactive forms of the enzyme, respectively, D2 the enzymatic complex, Q and C2 the fructose IP and fructose 2P, respectively. In the conformational equilibrium step (C2 I D3 <= D,) FDP activates the enzyme. Experimental results indicate that under physiological conditions, the enzyme is controlled mainly by the ATP and ADP. 

Second, GSH functions, presumably nonenzymically, in the reduction of protein thiols which have become oxidized to mixed disulfides (803). In this latter function GSH in some cases converts inactive enzymes to active ones, or vice versa, and may thus serve as a means of metabolic control. Examples of this important possibility are glycogen synthetase D (EC 2.4.1.11) and fructose-1,6-diphosphatase (EC 3.1.3.11). The D form of glycogen synthetase is dependent for activity upon the presence of glucose 6-phosphate. The enzyme is inactivated by GSSG and reactivated by GSH (204). Mixed disulfide formation between thiols of the enzyme and GSSG leads to a decrease in affinity of the enzyme for its activator (205). 

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