- Sucrose phosphorylase
Sucrose phosphorylase (E.C. 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 ofhexosyltransferases , a type ofglycosyltransferase that catalyzes the transfer of amonosaccharide from a phosphorylated sugar molecule to an acceptor molecule. More specifically,sucrose phosphorylase catalyzes the conversion ofsucrose to D-fructose and alpha-D-glucose-1-phosphate (Reid and Abratt 2005). It has been shown in multiple experiments that the enzyme catalyzes this conversion by aping-pong mechanism.Reaction
The method by which
sucrose phosphorylase convertssucrose to D-fructose and alpha-D-glucose-1-phosphate has been studied in great detail. In the reaction,sucrose is first phosphorylated, at which pointfructose is released by the enzyme-substrate complex. A covalent glucose-enzyme complex results, with beta-linkage between an oxygen atom in thepeptide bond and C-1 of glucose. The covalent complex was experimentally isolated by chemical modification of the protein using NaIO4 after addition of thesubstrate (Mirza et al. 2006), supporting the hypothesis that reaction catalyzed bysucrose phosphorylase proceeds through theping-pong mechanism. In the final enzymatic step, theglycosidic bond is hydrolyzed through the promotion of aphosphate group, yielding alpha-D-glucose-1-phosphate.In a separate reaction, alpha-D-glucose-1-phosphate is converted to
glucose-6-phosphate by the action ofphosphoglucomutase (Tedokon et al. 1992).Glucose-6-phosphate is an extremely important intermediate for several pathways in the human body, includingglycolysis ,gluconeogenesis , and thepentose phosphate pathway (Nelson and Cox 2005). The function ofsucrose phosphorylase is especially significant due to the role alpha-D-glucose-1-phosphate in energy metabolism.Structural Implications
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 (Sprogoe et al. 2004). The size of the enzyme, as determined by sedimentationcentrifugation , was found to be 55 KDa, consisting of 488 amino acids (Koga et al. 1991). The active has been shown to contain two binding sites, one designated a water site where hydroxylic molecules such as 1,2-cyclohexanediol andethylene 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 theglycosidic 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” (Schwarz and Nidetzky 2006). In fact,in vitro manipulation has shown that D-xylose, L-sorbose, and L-arabinose can replacefructose as the glucosyl acceptor (Mieyal, Simon, and Abeles 1972). The only requirement of the acceptor molecule is that the hydroxyl group on the C-3 be cis-disposed to the oxygen atom of theglycosidic bond . Glu-232 acts as the Bronsted acid-base catalyst, donating a proton to the displaced hydroxyl group on C-1 of the glucoside (Schwarz, Brecker, and Nidetsky 2007).The most significant residue in the enzymatic activity, however, is Asp-295 (Mueller and Nidetsky 2007). 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 (Mueller and Nidetsky 2007). This interaction is maximized during thetransition state of this covalent complex, lending support to theping-pong mechanism. Finally, phosphorylation of the glucosyl residue at C-1 forms a transient positive charge on the glucosyl carbon, promoting breakage of theester bond between Asp-192 and the sugar residue (Schwarz and Nidetsky 2006). 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 aretranscription al, affecting the amount of enzyme present at any given time.Global regulation of DNA molecules containing the gene for
sucrose phosphorylase is performed by [repression|catabolite repression}. First discovered inGram-negative bacteria, bothCyclic AMP (cAMP) and cAMP Receptor Protein (CRP) function insucrose phosphorylase regulation (Reid and Abratt 2005). The cAMP-CRP complex formed when both molecules combine acts as a positive regulator fortranscription of thesucrose phosphorylase gene. The complex binds to thepromoter region to activatetranscription , enhancing the creation ofsucrose phosphorylase (Nelson and Cox 2005).Genetic regulation of
sucrose phosphorylase is also performed bymetabolites . Through experimentation it is known that genes encoding for thesucrose phosphorylase enzyme can be induced bysucrose andraffinose (Trindade, Abratt, and Reid 2003).Glucose , on the other hand, represses thetranscription of thesucrose phosphorylase gene (Trindade, Abratt, and Reid 2003). Thesemetabolites undoubtedly function in this way because of their implications in cellular metabolism.There has been little research on methods of the
allosteric regulation ofsucrose 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 thatsucrose phosphorylase is additionally regulated by other commonmetabolites . For example, the presence of ATP would probably inhibitsucrose phosphorylase since ATP is a product of the catabolic pathway. Conversely, ADP would likely stimulatesucrose phosphorylase to increase levels of ATP. Further research on the subject would be required to support or refute these ideas.Metabolic Function
As mentioned above,
sucrose phosphorylase is a very important enzyme inmetabolism . The reaction catalyzed bysucrose phosphorylase produces the valuable byproducts alpha-D-glucose-1-phosphate andfructose . alpha-D-glucose-1-phosphate can be reversibly converted byphosphoglucomutase toglucose-6-phosphate (Tedokon et al. 1992), which is an important intermediate used inglycolysis . In addition,fructose can be reversibly converted intofructose-6-phosphate (Reid and Abratt 2005), also found in the glycolytic pathway. In fact,fructose-6-phosphate andglucose-6-phosphate can be interconverted in the glycolytic pathway byphosphohexose isomerase (Nelson and Cox 2005). The final product ofglycolysis ,pyruvate , has multiple implications inmetabolism . Duringanaerobic conditions,pyruvate con be converted into either lactate orethanol , depending on the organism, providing a quick source of energy. Inaerobic conditions,pyruvate can be converted intoAcetyl-CoA , which has many possible fates includingcatabolism in theCitric Acid Cycle for energy use andanabolism in the formation offatty 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 enhancestranscription of thesucrose phosphorylase gene (Reid and Abratt 2003) is only present whenglucose levels are low. The purpose ofsucrose phosphorylase , therefore, can be linked to the need for higherglucose levels, created through its reaction. The fact thatglucose acts as a feedback inhibitor to prevent the formation ofsucrose phosphorylase (Reid and Abratt 2005) further supports its catalytic role in the creation ofglucose for energy use or storage.The
glucose-6-phosphate molecule created from the original alpha-D-glucose-1-phosphate product is also involved in thepentose phosphate pathway . Through a series of reactions,glucose-6-phosphate can be converted intoribose 5-phosphate , which is used for a variety of molecules such asnucleotides ,coenzymes ,DNA , andRNA (Nelson and Cox 2005). These connections reveal thatsucrose phosphorylase is also important for the regulation of other cellular molecules.References:
# Koga, T., K. Nakamura, Y. Shirokane, K. Mizusawa, S. Kitao, M. Kikuchi. “Purification and some properties of sucrose phosphorylase from Leuconostoc mesenteroides.” "
Journal of Agricultural Biological Chemistry " 55.7 (1991): 1805-1810.
# Mietal, J.J., M. Simon, and R.H. Abeles. “Mechanism of action of sucrose phosphorylase.” "Journal of Biological Chemistry " 247.2 (1972): 532-542.
# Mirza, O., L.K. Skov, D. Sprongoe, L.A. van den Broek, G. Beldman, J.S. Kastrup, M. Gajhede. “Structural Rearrangements of Sucrose Phosphorylase from Bifidobacterium adolescentis during Sucrose Conversion.” "Journal of Biological Chemistry" 281.46 (2006): 35576-35584.
# Mueller, M., and B. Nidetzky. “The role of Asp-295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site-directed mutagenesis.” "FEBS Letter Journal " 581.7 (2007): 1403-1408.
# Nelson and Cox. "Lehninger Principles of Biochemistry." 4th ed. New York, W.H. Freeman and Company: 2005.
# Reid, Sharon J. and Valerie R. Abratt. “Sucrose utilization in bacteria: genetic organization and regulation.” "Journal of Applied Microbiology & Biotechnology " 67.3 (2005): 312-321.
# Schwarz, A., L. Brecker, and B. Nidetsky. “Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237 à Gln mutant enzymes.” "Biochemistry Journal " 403.3 (2007): 441-449.
# Schwarz, A. and B. Nidetzky. “Asp-196-->Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate.” "FEBS Letter Journal" 580.16 (2006): 3905-3910.
# Sprogoe, D., L.A. van den Broek, O. Mirza, J.S. Kastrup, A.G. Voragen, M. Gajhede, L.K. Skov. “Crystal Structure of Sucrose Phosphorylase from Bifidobacterium adolescentis.” "Biochemistry Journal" 43.5 (2004): 1156-62.
# Tedokon, M., K. Suzuki, Y. Kayamori, S. Fukita, and Y. Katayama. “Enzymatic assay of inorganic phosphate with use of sucrose phosphorylase and phosphoglucomutase.” "Journal of Clinical Chemistry " 38.4 (1992): 512-515.
# Trindade M.I., V. R. Abratt, and S.J. Reid. “Induction of the sucrose utilisation genes from Bifidobacterium lactis by sucrose and raffinose. “ "Journal of Applied Environmental Microbiology " 69 (2003): 24–32.External links
* [http://www.jbc.org/cgi/content/abstract/245/5/1020 The Mechanism of Action of Sucrose Phosphorylase]
* [http://www.brenda.uni-koeln.de/index.php4?page=information/all_enzymes.php4?ecno=2.4.1.7 BRENDA Sucrose Phosphorylase Reaction Mechanism]
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