Galactokinase Explained

Galactokinase 1
Caption:Cartoon structure of a human galactokinase 1 monomer in complex with galactose (red) and an ATP analogue (orange). A magnesium ion is visible as a green sphere. (From)
Hgncid:4118
Symbol:GALK1
Altsymbols:GALK
Entrezgene:2584
Omim:604313
Refseq:NM_000154
Uniprot:P51570
Ecnumber:2.7.1.6
Chromosome:17
Arm:q
Band:23
Locussupplementarydata:-q25
Galactokinase 2
Hgncid:4119
Symbol:GALK2
Entrezgene:2585
Omim:137028
Refseq:NM_002044
Uniprot:Q01415
Ecnumber:2.7.1.6
Chromosome:15

Galactokinase is an enzyme (phosphotransferase) that facilitates the phosphorylation of α-D-galactose to galactose 1-phosphate at the expense of one molecule of ATP.[1] Galactokinase catalyzes the second step of the Leloir pathway, a metabolic pathway found in most organisms for the catabolism of α-D-galactose to glucose 1-phosphate. First isolated from mammalian liver, galactokinase has been studied extensively in yeast, archaea,[2] plants,[3] [4] and humans.[5]

Structure

Galactokinase is composed of two domains separated by a large cleft. The two regions are known as the N- and C-terminal domains, and the adenine ring of ATP binds in a hydrophobic pocket located at their interface. The N-terminal domain is marked by five strands of mixed beta-sheet and five alpha-helices, and the C-terminal domain is characterized by two layers of anti-parallel beta-sheets and six alpha-helices. Galactokinase does not belong to the sugar kinase family, but rather to a class of ATP-dependent enzymes known as the GHMP superfamily.[6] GHMP is an abbreviation referring to its original members: galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase. Members of the GHMP superfamily have great three-dimensional similarity despite only ten to 20% sequence identity. These enzymes contain three well-conserved motifs (I, II, and III), the second of which is involved in nucleotide binding and has the sequence Pro-X-X-X-Gly-Leu-X-Ser-Ser-Ala.[7]

Sugar specificity

Galactokinases across different species display a great diversity of substrate specificities. E. coli galactokinase can also phosphorylate 2-deoxy-D-galactose, 2-amino-deoxy-D-galactose, 3-deoxy-D-galactose and D-fucose. The enzyme cannot tolerate any C-4 modifications, but changes at the C-2 position of D-galactose do not interfere with enzyme function.[8] Both human and rat galactokinases are also able to successfully phosphorylate 2-deoxy-D-galactose.[9] [10] Galactokinase from S. cerevisiae, on the other hand, is highly specific for D-galactose and cannot phosphorylate glucose, mannose, arabinose, fucose, lactose, galactitol, or 2-deoxy-D-galactose.[11] [12] Moreover, the kinetic properties of galactokinase also differ across species. The sugar specificity of galactokinases from different sources has been dramatically expanded through directed evolution[13] and structure-based protein engineering.[14] [15] The corresponding broadly permissive sugar anomeric kinases serve as a cornerstone for in vitro and in vivo glycorandomization.[16] [17] [18]

Mechanism

Recently, the roles of active site residues in human galactokinase have become understood. Asp-186 abstracts a proton from C1-OH of α-D-galactose, and the resulting alkoxide nucleophile attacks the γ-phosphorus of ATP. A phosphate group is transferred to the sugar, and Asp-186 may be deprotonated by water. Nearby Arg-37 stabilizes Asp-186 in its anionic form and has also been proven to be essential to galactokinase function in point mutation experiments.[19] Both the aspartic acid and arginine active site residues are highly conserved among galactokinases.

Biological function

The Leloir pathway catalyzes the conversion of galactose to glucose. Galactose is found in dairy products, as well as in fruits and vegetables, and can be produced endogenously in the breakdown of glycoproteins and glycolipids. Three enzymes are required in the Leloir pathway: galactokinase, galactose-1-phosphate uridylyltransferase, and UDP-galactose 4-epimerase. Galactokinase catalyzes the first committed step of galactose catabolism, forming galactose 1-phosphate.[20]

Disease relevance

Galactosemia, a rare metabolic disorder characterized by decreased ability to metabolize galactose, can be caused by a mutation in any of the three enzymes in the Leloir pathway.[21] Galactokinase deficiency, also known as galactosemia type II, is a recessive metabolic disorder caused by a mutation in human galactokinase. About 20 mutations have been identified that cause galactosemia type II, the main symptom of which is early onset cataracts. In lens cells of the human eye, aldose reductase converts galactose to galactitol. As galactose is not being catabolized to glucose due to a galactokinase mutation, galactitol accumulates. This galactitol gradient across the lens cell membrane triggers the osmotic uptake of water, and the swelling and eventual apoptosis of lens cells ensues.[22]

Notes and References

  1. Web site: galactokinase . Medical Dictionary . 2013-01-26.
  2. Hartley A, Glynn SE, Barynin V, Baker PJ, Sedelnikova SE, Verhees C, de Geus D, van der Oost J, Timson DJ, Reece RJ, Rice DW . Substrate specificity and mechanism from the structure of Pyrococcus furiosus galactokinase . Journal of Molecular Biology . 337 . 2 . 387–98 . March 2004 . 15003454 . 10.1016/j.jmb.2004.01.043 .
  3. Foglietti MJ, Percheron F . [Purification and mechanism of action of a plant galactokinase] . Biochimie . 58 . 5 . 499–504 . 1976 . 182286 . 10.1016/s0300-9084(76)80218-0 .
  4. Dey PM . Galactokinase of Vicia faba seeds . European Journal of Biochemistry . 136 . 1 . 155–9 . October 1983 . 6617655 . 10.1111/j.1432-1033.1983.tb07720.x . free .
  5. Holden HM, Thoden JB, Timson DJ, Reece RJ . Galactokinase: structure, function and role in type II galactosemia . Cellular and Molecular Life Sciences . 61 . 19–20 . 2471–84 . October 2004 . 15526155 . 10.1007/s00018-004-4160-6 . 7293337 .
  6. Tang M, Wierenga K, Elsas LJ, Lai K . Molecular and biochemical characterization of human galactokinase and its small molecule inhibitors . Chemico-Biological Interactions . 188 . 3 . 376–85 . December 2010 . 20696150 . 2980576 . 10.1016/j.cbi.2010.07.025 . 2010CBI...188..376T .
  7. Thoden JB, Holden HM . Molecular structure of galactokinase . The Journal of Biological Chemistry . 278 . 35 . 33305–11 . August 2003 . 12796487 . 10.1074/jbc.M304789200 . free .
  8. Yang J, Fu X, Jia Q, Shen J, Biggins JB, Jiang J, Zhao J, Schmidt JJ, Wang PG, Thorson JS . Studies on the substrate specificity of Escherichia coli galactokinase . Organic Letters . 5 . 13 . 2223–6 . June 2003 . 12816414 . 10.1021/ol034642d .
  9. Timson DJ, Reece RJ . Sugar recognition by human galactokinase . BMC Biochemistry . 4 . 16 . November 2003 . 14596685 . 280648 . 10.1186/1471-2091-4-16 . free .
  10. Walker DG, Khan HH . Some properties of galactokinase in developing rat liver . The Biochemical Journal . 108 . 2 . 169–75 . June 1968 . 5665881 . 1198790 . 10.1042/bj1080169.
  11. Schell MA, Wilson DB . Purification of galactokinase mRNA from Saccharomyces cerevisiae by indirect immunoprecipitation . The Journal of Biological Chemistry . 254 . 9 . 3531–6 . May 1979 . 10.1016/S0021-9258(18)50793-6 . 107173 . free .
  12. Sellick CA, Reece RJ . Contribution of amino acid side chains to sugar binding specificity in a galactokinase, Gal1p, and a transcriptional inducer, Gal3p . The Journal of Biological Chemistry . 281 . 25 . 17150–5 . June 2006 . 16603548 . 10.1074/jbc.M602086200 . free.
  13. Hoffmeister D, Yang J, Liu L, Thorson JS . Creation of the first anomeric D/L-sugar kinase by means of directed evolution . Proceedings of the National Academy of Sciences of the United States of America . 100 . 23 . 13184–9 . November 2003 . 14612558 . 263743 . 10.1073/pnas.2235011100 . free .
  14. Yang J, Fu X, Liao J, Liu L, Thorson JS . Structure-based engineering of E. coli galactokinase as a first step toward in vivo glycorandomization . Chemistry & Biology . 12 . 6 . 657–64 . June 2005 . 15975511 . 10.1016/j.chembiol.2005.04.009 . free .
  15. Williams GJ, Gantt RW, Thorson JS . The impact of enzyme engineering upon natural product glycodiversification . Current Opinion in Chemical Biology . 12 . 5 . 556–64 . October 2008 . 18678278 . 4552347 . 10.1016/j.cbpa.2008.07.013 .
  16. Langenhan JM, Griffith BR, Thorson JS . Neoglycorandomization and chemoenzymatic glycorandomization: two complementary tools for natural product diversification . Journal of Natural Products . 68 . 11 . 1696–711 . November 2005 . 16309329 . 10.1021/np0502084 .
  17. Williams GJ, Yang J, Zhang C, Thorson JS . Recombinant E. coli prototype strains for in vivo glycorandomization . ACS Chemical Biology . 6 . 1 . 95–100 . January 2011 . 20886903 . 3025069 . 10.1021/cb100267k .
  18. Gantt RW, Peltier-Pain P, Thorson JS . Enzymatic methods for glyco(diversification/randomization) of drugs and small molecules . Natural Product Reports . 28 . 11 . 1811–53 . October 2011 . 21901218 . 10.1039/c1np00045d .
  19. Megarity CF, Huang M, Warnock C, Timson DJ . The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases . Bioorganic Chemistry . 39 . 3 . 120–6 . June 2011 . 21474160. 10.1016/j.bioorg.2011.03.001 .
  20. Holden HM, Rayment I, Thoden JB . Structure and function of enzymes of the Leloir pathway for galactose metabolism . The Journal of Biological Chemistry . 278 . 45 . 43885–8 . November 2003 . 12923184 . 10.1074/jbc.R300025200 . free .
  21. Frey PA . The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose . FASEB Journal . 10 . 4 . 461–70 . March 1996 . 8647345 . 10.1096/fasebj.10.4.8647345. free . 13857006 .
  22. Timson DJ, Reece RJ . Functional analysis of disease-causing mutations in human galactokinase . European Journal of Biochemistry . 270 . 8 . 1767–74 . April 2003 . 12694189 . 10.1046/j.1432-1033.2003.03538.x . free .

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