Sucrose phosphorylase

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Sucrose phosphorylase
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Sucrose phosphorylase homodimer, Bifidobacterium adolescentis
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EC no. 2.4.1.7
CAS no. 9074-06-0
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Sucrose phosphorylase (EC 2.4.1.7) is an important enzyme in the metabolism of sucrose and regulation of other metabolic intermediates. Sucrose phosphorylase is in the class of hexosyltransferases. More specifically it has been placed in the retaining glycoside hydrolases family although it catalyzes a transglycosidation rather than hydrolysis. Sucrose phosphorylase catalyzes the conversion of sucrose to D-fructose and α-D-glucose-1-phosphate. [1] It has been shown in multiple experiments that the enzyme catalyzes this conversion by a double displacement mechanism.

Contents

Reaction

The method by which sucrose phosphorylase converts sucrose to D-fructose and alpha-D-glucose-1-phosphate has been studied in great detail. In the reaction, sucrose binds to the enzyme, at which point fructose is released by the enzyme-substrate complex. A covalent glucose-enzyme complex results, with beta-linkage between an oxygen atom in the carboxyl group of an aspartyl residue and C-1 of glucose. The covalent complex was experimentally isolated by chemical modification of the protein using NaIO4 after addition of the substrate, [2] [3] supporting the hypothesis that reaction catalyzed by sucrose phosphorylase proceeds through the ping-pong mechanism. In the final enzymatic step, the glycosidic bond is cleaved through reaction with a phosphate group, yielding α-D-glucose-1-phosphate.

In a separate reaction, α-D-glucose-1-phosphate is converted to glucose-6-phosphate by the action of phosphoglucomutase. [4] Glucose-6-phosphate is an extremely important intermediate for several pathways in the human body, including glycolysis, gluconeogenesis, and the pentose phosphate pathway. [5] The function of sucrose phosphorylase is especially significant due to the role α-D-glucose-1-phosphate in energy metabolism.

Structure

The structure of sucrose phosphorylase has been identified in numerous experiments. The enzyme consists of four major domains, namely A, B, B’, and C. Domains A, B’ and C exist as dimers around the active site. [6] The size of the enzyme, as determined by sedimentation centrifugation, was found to be 55 KDa, consisting of 488 amino acids. [7] The active has been shown to contain two binding sites, one designated a water site where hydroxylic molecules such as 1,2-cyclohexanediol and ethylene glycol may bind, and another designated as the acceptor site where the sugar molecule binds. Though the function of the water site has not been completely elucidated, the enzyme's stability in aqueous solutions indicates that the water site may be involved in hydrolysis of the glycosidic bond.

The acceptor site is surrounded by three active residues that have been found to be essential in enzymatic activity. Using specific mutagenic assays, Asp-192 was found to be the catalytic nucleophile of the enzyme, “attacking C-1 of the glucosyl moiety of sucrose”. [8] In fact, in vitro manipulation has shown that D-xylose, L-sorbose, and L-arabinose can replace fructose as the glucosyl acceptor. [9] The only requirement of the acceptor molecule is that the hydroxyl group on the C-3 be cis-disposed to the oxygen atom of the glycosidic bond. Glu-232 acts as the Bronsted acid-base catalyst, donating a proton to the displaced hydroxyl group on C-1 of the glucoside. [10]

The most significant residue in the enzymatic activity, however, is Asp-295. [11] Upon cleavage of the fructofuranosyl moiety from sucrose, the resultant glucose forms a covalent intermediate with the enzyme. The carboxylate side chain of Asp-295 hydrogen bonds with the hydroxyl groups at C-2 and C-3 of the glucosyl residue. [11] This interaction is maximized during the transition state of this covalent complex, lending support to the ping-pong mechanism. Finally, phosphorylation of the glucosyl residue at C-1 forms a transient positive charge on the glucosyl carbon, promoting breakage of the ester bond between Asp-192 and the sugar residue. [8] Cleavage yields the product, α-D-glucose-1-phosphate.

Regulation

Since the discovery and characterization of sucrose phosphorylase, few documented experiments discuss mechanisms of regulation for the enzyme. The known methods of regulation are transcriptional, affecting the amount of enzyme present at any given time.

Global regulation of DNA molecules containing the gene for sucrose phosphorylase is performed by catabolite repression. First discovered in Gram-negative bacteria, both Cyclic AMP (cAMP) and cAMP Receptor Protein (CRP) function in sucrose phosphorylase regulation. [1] The cAMP-CRP complex formed when both molecules combine acts as a positive regulator for transcription of the sucrose phosphorylase gene. The complex binds to the promoter region to activate transcription, enhancing the creation of sucrose phosphorylase. [5]

Genetic regulation of sucrose phosphorylase is also performed by metabolites. Through experimentation it is known that genes encoding for the sucrose phosphorylase enzyme can be induced by sucrose and raffinose. [12] Glucose, on the other hand, represses the transcription of the sucrose phosphorylase gene. [12] These metabolites undoubtedly function in this way because of their implications in cellular metabolism.

There has been little research on methods of the allosteric regulation of sucrose phosphorylase, so at this point the function of allosteric molecules can only be hypothesized. Due to the nature of its function in metabolic pathways, it is likely that sucrose phosphorylase is additionally regulated by other common metabolites.[ citation needed ] For example, the presence of ATP would probably inhibit sucrose phosphorylase since ATP is a product of the catabolic pathway. Conversely, ADP would likely stimulate sucrose phosphorylase to increase levels of ATP. Further research on the subject would be required to support or refute these ideas.

Function

As mentioned above, sucrose phosphorylase is a very important enzyme in metabolism. The reaction catalyzed by sucrose phosphorylase produces the valuable byproducts α-D-glucose-1-phosphate and fructose. α-D-glucose-1-phosphate can be reversibly converted by phosphoglucomutase to glucose-6-phosphate, [4] which is an important intermediate used in glycolysis. In addition, fructose can be reversibly converted into fructose 6-phosphate, [1] also found in the glycolytic pathway. In fact, fructose-6-phosphate and glucose-6-phosphate can be interconverted in the glycolytic pathway by phosphohexose isomerase. [5] The final product of glycolysis, pyruvate, has multiple implications in metabolism. During anaerobic conditions, pyruvate con be converted into either lactate or ethanol, depending on the organism, providing a quick source of energy. In aerobic conditions, pyruvate can be converted into Acetyl-CoA, which has many possible fates including catabolism in the Citric Acid Cycle for energy use and anabolism in the formation of fatty acids for energy storage. Through these reactions, sucrose phosphorylase becomes important in the regulation of metabolic functions.

The regulation of sucrose phosphorylase can also be used to explain its function in terms of energy consumption and preservation. The cAMP-CRP complex that enhances transcription of the sucrose phosphorylase gene (Reid and Abratt 2003) is only present when glucose levels are low. The purpose of sucrose phosphorylase, therefore, can be linked to the need for higher glucose levels, created through its reaction. The fact that glucose acts as a feedback inhibitor to prevent the formation of sucrose phosphorylase [1] further supports its catalytic role in the creation of glucose for energy use or storage.

The glucose-6-phosphate molecule created from the original α-D-glucose-1-phosphate product is also involved in the pentose phosphate pathway. Through a series of reactions, glucose-6-phosphate can be converted into ribose-5-phosphate, which is used for a variety of molecules such as nucleotides, coenzymes, DNA, and RNA. [5] These connections reveal that sucrose phosphorylase is also important for the regulation of other cellular molecules.

Related Research Articles

<span class="mw-page-title-main">Glycolysis</span> Series of interconnected biochemical reactions

Glycolysis is the metabolic pathway that converts glucose into pyruvate and, in most organisms, occurs in the liquid part of cells. The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH). Glycolysis is a sequence of ten reactions catalyzed by enzymes.

<span class="mw-page-title-main">Kinase</span> Enzyme catalyzing transfer of phosphate groups onto specific substrates

In biochemistry, a kinase is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule donates a phosphate group to the substrate molecule. This transesterification produces a phosphorylated substrate and ADP. Conversely, it is referred to as dephosphorylation when the phosphorylated substrate donates a phosphate group and ADP gains a phosphate group. These two processes, phosphorylation and dephosphorylation, occur four times during glycolysis.

<span class="mw-page-title-main">Glucagon</span> Peptide hormone

Glucagon is a peptide hormone, produced by alpha cells of the pancreas. It raises the concentration of glucose and fatty acids in the bloodstream and is considered to be the main catabolic hormone of the body. It is also used as a medication to treat a number of health conditions. Its effect is opposite to that of insulin, which lowers extracellular glucose. It is produced from proglucagon, encoded by the GCG gene.

<span class="mw-page-title-main">Glycogenolysis</span> Breakdown of glycogen

Glycogenolysis is the breakdown of glycogen (n) to glucose-1-phosphate and glycogen (n-1). Glycogen branches are catabolized by the sequential removal of glucose monomers via phosphorolysis, by the enzyme glycogen phosphorylase.

<span class="mw-page-title-main">Phosphofructokinase 1</span> Class of enzymes

Phosphofructokinase-1 (PFK-1) is one of the most important regulatory enzymes of glycolysis. It is an allosteric enzyme made of 4 subunits and controlled by many activators and inhibitors. PFK-1 catalyzes the important "committed" step of glycolysis, the conversion of fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP. Glycolysis is the foundation for respiration, both anaerobic and aerobic. Because phosphofructokinase (PFK) catalyzes the ATP-dependent phosphorylation to convert fructose-6-phosphate into fructose 1,6-bisphosphate and ADP, it is one of the key regulatory steps of glycolysis. PFK is able to regulate glycolysis through allosteric inhibition, and in this way, the cell can increase or decrease the rate of glycolysis in response to the cell's energy requirements. For example, a high ratio of ATP to ADP will inhibit PFK and glycolysis. The key difference between the regulation of PFK in eukaryotes and prokaryotes is that in eukaryotes PFK is activated by fructose 2,6-bisphosphate. The purpose of fructose 2,6-bisphosphate is to supersede ATP inhibition, thus allowing eukaryotes to have greater sensitivity to regulation by hormones like glucagon and insulin.

<span class="mw-page-title-main">Phosphoglucomutase</span>

Phosphoglucomutase is an enzyme that transfers a phosphate group on an α-D-glucose monomer from the 1 to the 6 position in the forward direction or the 6 to the 1 position in the reverse direction.

<span class="mw-page-title-main">Phosphatase</span> Enzyme which catalyzes the removal of a phosphate group from a molecule

In biochemistry, a phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol. Because a phosphatase enzyme catalyzes the hydrolysis of its substrate, it is a subcategory of hydrolases. Phosphatase enzymes are essential to many biological functions, because phosphorylation and dephosphorylation serve diverse roles in cellular regulation and signaling. Whereas phosphatases remove phosphate groups from molecules, kinases catalyze the transfer of phosphate groups to molecules from ATP. Together, kinases and phosphatases direct a form of post-translational modification that is essential to the cell's regulatory network.

<span class="mw-page-title-main">Glucose 6-phosphate</span> Chemical compound

Glucose 6-phosphate is a glucose sugar phosphorylated at the hydroxy group on carbon 6. This dianion is very common in cells as the majority of glucose entering a cell will become phosphorylated in this way.

In biochemistry, isomerases are a general class of enzymes that convert a molecule from one isomer to another. Isomerases facilitate intramolecular rearrangements in which bonds are broken and formed. The general form of such a reaction is as follows:

Glycogenesis is the process of glycogen synthesis, in which glucose molecules are added to chains of glycogen for storage. This process is activated during rest periods following the Cori cycle, in the liver, and also activated by insulin in response to high glucose levels.

<span class="mw-page-title-main">Glycogen phosphorylase</span> Class of enzymes

Glycogen phosphorylase is one of the phosphorylase enzymes. Glycogen phosphorylase catalyzes the rate-limiting step in glycogenolysis in animals by releasing glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Glycogen phosphorylase is also studied as a model protein regulated by both reversible phosphorylation and allosteric effects.

<span class="mw-page-title-main">Glucose 1-phosphate</span> Chemical compound

Glucose 1-phosphate is a glucose molecule with a phosphate group on the 1'-carbon. It can exist in either the α- or β-anomeric form.

<span class="mw-page-title-main">Glycogen synthase</span> Enzyme class, includes all types of glycogen/starch synthases

Glycogen synthase is a key enzyme in glycogenesis, the conversion of glucose into glycogen. It is a glycosyltransferase that catalyses the reaction of UDP-glucose and n to yield UDP and n+1.

Glucose-1,6-bisphosphate synthase is a type of enzyme called a phosphotransferase and is involved in mammalian starch and sucrose metabolism. It catalyzes the transfer of a phosphate group from 1,3-bisphosphoglycerate to glucose-1-phosphate, yielding 3-phosphoglycerate and glucose-1,6-bisphosphate.

In enzymology, an alpha,alpha-trehalose phosphorylase (configuration-retaining) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Alternansucrase</span> Enzyme

In enzymology, an alternansucrase is an enzyme that catalyzes a chemical reaction that transfers an alpha-D-glucosyl residue from sucrose alternately to the 6- and 3-positions of the non-reducing terminal residue of an alpha-D-glucan, thereby creating a glucan with alternating alpha-1,6- and alpha-1,3-bonds. The name "alternan" was coined in 1982 for the glucan based on its alternating linkage structure.

<span class="mw-page-title-main">Sucrose-phosphate synthase</span> Class of enzymes

Sucrose-phosphate synthase (SPS) is a plant enzyme involved in sucrose biosynthesis. Specifically, this enzyme catalyzes the transfer of a hexosyl group from uridine diphosphate glucose (UDP-glucose) to D-fructose 6-phosphate to form UDP and D-sucrose-6-phosphate. This reversible step acts as the key regulatory control point in sucrose biosynthesis, and is an excellent example of various key enzyme regulation strategies such as allosteric control and reversible phosphorylation.

<span class="mw-page-title-main">Inborn errors of carbohydrate metabolism</span> Medical condition

Inborn errors of carbohydrate metabolism are inborn error of metabolism that affect the catabolism and anabolism of carbohydrates.

<span class="mw-page-title-main">Glucansucrase</span> Enzyme

Glucansucrase is an enzyme in the glycoside hydrolase family GH70 used by lactic acid bacteria to split sucrose and use resulting glucose molecules to build long, sticky biofilm chains. These extracellular homopolysaccharides are called α-glucan polymers.

Maltodextrin phosphorylase is a phosphorylase enzyme, more specifically one type of glycosyltransferase. Maltodextrin phosphorylase plays a critical role in maltodextrin metabolism in E. coli. This bacterial enzyme, often referred to as MalP, catalyzes the phosphorolysis of an α-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). Phosphorylases are well-regarded for their allosteric effects on metabolism, however MalP exhibits no allosteric properties. It has a higher affinity for linear oligosaccharides than the related glycogen phosphorylase.

References

  1. 1 2 3 4 Reid SJ, Abratt VR (May 2005). "Sucrose utilisation in bacteria: genetic organisation and regulation". Applied Microbiology and Biotechnology. 67 (3): 312–21. doi:10.1007/s00253-004-1885-y. PMID   15660210.
  2. ↑ Voet JG, Abeles RH (March 1970). "The mechanism of action of sucrose phosphorylase. Isolation and properties of a beta-linked covalent glucose-enzyme complex". The Journal of Biological Chemistry. 245 (5): 1020–31. PMID   4313700.
  3. ↑ Mirza O, Skov LK, Sprogøe D, van den Broek LA, Beldman G, Kastrup JS, Gajhede M (November 2006). "Structural rearrangements of sucrose phosphorylase from Bifidobacterium adolescentis during sucrose conversion". The Journal of Biological Chemistry. 281 (46): 35576–84. doi: 10.1074/jbc.M605611200 . PMID   16990265.
  4. 1 2 Tedokon M, Suzuki K, Kayamori Y, Fujita S, Katayama Y (April 1992). "Enzymatic assay of inorganic phosphate with use of sucrose phosphorylase and phosphoglucomutase". Clinical Chemistry. 38 (4): 512–5. PMID   1533182.
  5. 1 2 3 4 Nelson DL, Cox MM (2005). Lehninger Principles of Biochemistry (4th ed.). New York: W.H. Freeman and Company.
  6. ↑ Sprogøe D, van den Broek LA, Mirza O, Kastrup JS, Voragen AG, Gajhede M, Skov LK (February 2004). "Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis". Biochemistry. 43 (5): 1156–62. doi:10.1021/bi0356395. PMID   14756551.
  7. ↑ Koga T, Nakamura K, Shirokane Y, Mizusawa K, Kitao S, Kikuchi M (July 1991). "Purification and some properties of sucrose phosphorylase from Leuconostoc mesenteroides". Agricultural and Biological Chemistry. 55 (7): 1805–10. PMID   1368718.
  8. 1 2 Schwarz A, Nidetzky B (July 2006). "Asp-196-->Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate". FEBS Letters. 580 (16): 3905–10. doi: 10.1016/j.febslet.2006.06.020 . PMID   16797542.
  9. ↑ Mieyal JJ, Simon M, Abeles RH (January 1972). "Mechanism of action of sucrose phosphorylase. 3. The reaction with water and other alcohols". The Journal of Biological Chemistry. 247 (2): 532–42. PMID   5009699.
  10. ↑ Schwarz A, Brecker L, Nidetzky B (May 2007). "Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237-->Gln mutant enzymes". The Biochemical Journal. 403 (3): 441–9. doi:10.1042/BJ20070042. PMC   1876375 . PMID   17233628.
  11. 1 2 Mueller M, Nidetzky B (April 2007). "The role of Asp-295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site-directed mutagenesis". FEBS Letters. 581 (7): 1403–8. doi:10.1016/j.febslet.2007.02.060. PMID   17350620.
  12. 1 2 Trindade MI, Abratt VR, Reid SJ (January 2003). "Induction of sucrose utilization genes from Bifidobacterium lactis by sucrose and raffinose". Applied and Environmental Microbiology. 69 (1): 24–32. doi:10.1128/AEM.69.1.24-32.2003. PMC   152442 . PMID   12513973.
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