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Phosphoribulokinase

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phosphoribulokinase
3D cartoon depiction of a phosphoribulokinase protomer from Methanospirillum hungatei
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EC no.2.7.1.19
CAS no.9030-60-8
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Phosphoribulokinase (PRK)(EC2.7.1.19) is an essentialphotosyntheticenzymethatcatalyzestheATP-dependentphosphorylationofribulose 5-phosphate(RuP) intoribulose 1,5-bisphosphate(RuBP), bothintermediatesin theCalvin Cycle.Its main function is to regenerate RuBP, which is the initialsubstrateand CO2-acceptor molecule of the Calvin Cycle.[1]PRK belongs to the family oftransferase enzymes,specifically those transferring phosphorus-containing groups (phosphotransferases) to an alcohol group acceptor. Along withribulose 1,5-bisphosphate carboxylase/oxygenase(RuBisCo), phosphoribulokinase is unique to the Calvin Cycle.[2]Therefore, PRK activity often determines themetabolic ratein organisms for whichcarbon fixationis key to survival.[3]Much initial work on PRK was done withspinachleaf extracts in the 1950s; subsequent studies of PRK in other photosyntheticprokaryoticandeukaryoticorganisms have followed. The possibility that PRK might exist was first recognized by Weissbach et al. in 1954; for example, the group noted thatcarbon dioxidefixation in crude spinach extracts was enhanced by the addition of ATP.[3][4]The first purification of PRK was conducted by Hurwitz and colleagues in 1956.[5][6][7] 

ATP + Mg2+- D-ribulose 5-phosphateADP + D-ribulose 1,5-bisphosphate
Reaction scheme for the regeneration of ribulose 1,5-bisphosphate from ribulose 5-phosphate by phosphoribulokinase[1]

The twosubstratesof PRK areATPandD-ribulose 5-phosphate,whereas its twoproductsareADPandD-ribulose 1,5-bisphosphate.PRK activity requires the presence of adivalentmetalcationlike Mg2+,as indicated in the reaction above.[3]

Structure

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The structure of PRK is different in prokaryotes and eukaryotes. Prokaryotic PRK's typically exist asoctamersof 32 kDasubunits,while eukaryotic PRK's are oftendimersof 40 kDa subunits.[8][9]Structural determinations for eukaryotic PRK have yet to be conducted, but prokaryotic PRK structures are still useful for rationalizing the regulation and mechanism of PRK. As of 2018, only two crystal structures have been resolved for this class of enzymes inRhodobacter sphaeroidesandMethanospirillum hungatei,with the respectivePDBaccession codes1A7Jand5B3F.

Key residues that interact with RuP (labeled in blue) or with the hydroxyl group in RuP (red) within the active site ofR. sphaeroidesPRK. Generated from1A7J.Click to view enlarged.

Rhodobacter sphaeroides

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InRhodobacter sphaeroides,PRK (or RsPRK) exists as ahomooctomerwithprotomerscomposed of seven-strandedmixed β-sheets,sevenα-helices,and an auxiliary pair of anti-parallelβ-strands.[10]The RsPRK subunit exhibits aprotein foldinganalogous to the folding ofnucleotide monophosphate (NMP) kinases.[3]Mutagenesis studiessuggest that eitherAsp42 or Asp 169 acts as thecatalyticbasethatdeprotonatesthe O1hydroxyloxygen on RuP fornucleophilic attackof ATP, while the other acts aligandfor a metal cation likeMg2+(read mechanism below for more details).[10]Otherresiduespresent at theactive sitefor RsPRK includeHis45,Arg49, Arg 168, and Arg 173, which are purportedly involved in RuP binding.[10](See image at right).

Methanospirillum hungatei

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InarchaealPRK ofMethanospirillum hungatei,PRK (or MhPRK) exists as ahomodimerof twoprotomers,each consisting of eight-stranded mixed β-sheets surrounded by α-helices and β-strands—similar to the structure of bacterial PRK fromR. sphaeroides(see info. box above).[11]Although theirquaternary structuresdiffer and they have lowamino acid sequence identity,MhPRK and RsPRK have structurally similarN-terminal domainsas well as sequentially conserved residues like His 55,Lys151, and Arg 154.[11]

Mechanism and Activity

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PRK catalyzes the phosphorylation of RuP into RuBP. A catalytic residue in the enzyme (i.e. aspartate in RsPRK) deprotonates the O1 hydroxyl oxygen on RuP andactivatesit for nucleophilic attack of theγ-phosphoryl groupof ATP.[10]As the γ-phosphoryl group is transferred from ATP to RuP, itsstereochemistryinverts.[12]To allow for such inversion, the catalytic mechanism of PRK must not involve a phosphoryl-enzymeintermediate.[12]

Some studies suggest that both substrates (ATP and RuP) bind simultaneously to PRK and form aternary complex.Others suggest that the substrate addition is sequential; the particular order by which substrates are added is still disputed, and may in fact, vary for different organisms.[13][14]In addition to binding its substrates, PRK also requiresligationto divalent metal cations likeMg2+orMn2+for activity;Hg2+has been demonstrated to inactivate the enzyme.[3][15]

Enzyme specificity

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PRK shows highspecificityfor ribulose 5-phosphate. It does not act on any of the following substrates:D-xylulose 5-phosphate,fructose 6-phosphate,andsedoheptulose 7-phosphate.[15]However, at highconcentrations,PRK may sometimes phosphorylateribose 5-phosphate,a compound upstream theRuBP regeneration stepin the Calvin Cycle.[15]Furthermore, PRK isolated fromAlcaligenes eutrophushas been shown to useuridine triphosphate(UTP) andguanosine triphosphate(GTP) as alternative substrates to ATP.[8][3]

pH effects

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The phosphorylation reaction proceeds with maximalvelocityatpH7.9, with no detectable activity at pH's below 5.5 or above 9.0.[15]

Regulation

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Themechanismsby which prokaryotic and eukaryotic PRK's areregulatedvary. Prokaryotic PRK's are typically subject toallosteric regulationwhile eukaryotic PRK's are often regulated byreversiblethiol/disulfideexchange.[16]These differences are likely due to structural differences in theirC-terminal domains[11]

Allosteric regulation of prokaryotic PRK

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NADHis known to stimulate PRK activity, whileAMPandphosphoenolpyruvate(PEP) are known to inhibit activity.[3]AMP has been shown to be involved incompetitive inhibitioninThiobacillus ferrooxidansPRK.[17]On the other hand, PEP acts as anon-competitive inhibitorof PRK.[18]

Regulation of eukaryotic PRK

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Eukaryotic PRK is typically regulated through the reversibleoxidation/reductionof itscysteinesulfhydrylgroups, but studies suggests that its activity can be regulated by otherproteinsormetabolitesin thechloroplast.Of such metabolites,6-phosphogluconatehas been shown to be the most effective inhibitor of eukaryotic PRK by competing with RuP for the enzyme's active site.[19]This phenomenon may arise from the similarity inmolecular structurebetween 6-phosphogluconate and RuP.

More recent work on the regulation of eukaryotic PRK has focused on its ability to formmulti-enzyme complexeswith other Calvin cycle enzymes such asglyceraldehyde 3-phosphate dehydrogenase(G3PDH) or RuBisCo.[20]InChlamydomonas reinhardtii,chloroplast PRK and G3PDH exist as a bi-enzyme complex of 2 molecules of dimeric PRK and 2 molecules oftetramericG3PDH thorough association by an Arg 64 residue, which may potentially transfer information between the two enzymes as well.[21]

Multi-enzyme complexes are likely to have more intricate regulatory mechanisms, and studies have already probed such processes. For example, it has been shown that PRK-glyceraldehyde 3-phosphate dehydrogenase complexes inScenedesmus obliquusonly dissociate to release activated forms of its constituent enzymes in the presence ofNADPH,dithiothreitol (DTT),andthioredoxin.[22]Another topic of interest has been to compare the relative levels of PRK activity for when it is complexed to when it is not. For different photosynthetic eukaryotes, the enzyme activity of complexed PRK may be enhanced as opposed to free PRK, and vice versa.[23][24]

Other names

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Thesystematic nameof this enzyme class is ATP:D-ribulose-5-phosphate 1-phosphotransferase. Other names in common use include phosphopentokinase, ribulose-5-phosphate kinase, phosphopentokinase, phosphoribulokinase (phosphorylating), 5-phosphoribulose kinase, ribulose phosphate kinase, PKK, PRuK, and PRK.

References

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  1. ^abBerg, Jeremy M.; Tymoczko, John L.; Gatto, Jr., Gregory J.; Stryer, Lubert (2015-04-08).Biochemistry(Eighth ed.). New York.ISBN978-1464126109.OCLC913469736.{{cite book}}:CS1 maint: location missing publisher (link)
  2. ^Marsden WJ (September 16, 1983)."Purification and Molecular and Catalytic Properties of Phosphoribulokinase from the Cyanobacterium Chlorogloeopsis fritschii".Journal of General Microbiology.130(4): 999–1006.doi:10.1099/00221287-130-4-999.
  3. ^abcdefgMiziorko HM (2000). "Phosphoribulokinase: Current Perspectives on the Structure/Function Basis for Regulation and Catalysis". In Purich DL (ed.).Advances in Enzymology and Related Areas of Molecular Biology.Advances in Enzymology - and Related Areas of Molecular Biology. Vol. 74. John Wiley & Sons, Inc. pp. 95–127.doi:10.1002/9780470123201.ch3.ISBN9780470123201.PMID10800594.
  4. ^Weissbach A, Smyrniotis PZ, Horecker BL (July 1954). "Pentose phosphate and CO2 fixation with spinach extracts".Journal of the American Chemical Society.76(13): 3611–3612.doi:10.1021/ja01642a090.
  5. ^Hurwitz J, Weissbach A, Horecker BL, Smyrniotis PZ (February 1956)."Spinach phosphoribulokinase".The Journal of Biological Chemistry.218(2): 769–83.doi:10.1016/S0021-9258(18)65841-7.PMID13295229.
  6. ^Racker E (July 1957). "The reductive pentose phosphate cycle. I. Phosphoribulokinase and ribulose diphosphate carboxylase".Archives of Biochemistry and Biophysics.69:300–10.doi:10.1016/0003-9861(57)90496-4.PMID13445203.
  7. ^Jakoby WB, Brummond DO, Ochoa S (February 1956)."Formation of 3-phosphoglyceric acid by carbon dioxide fixation with spinach leaf enzymes".The Journal of Biological Chemistry.218(2): 811–22.doi:10.1016/S0021-9258(18)65844-2.PMID13295232.
  8. ^abSiebert K, Schobert P, Bowien B (March 1981). "Purification, some catalytic and molecular properties of phosphoribulokinase from Alcaligenes eutrophus".Biochimica et Biophysica Acta (BBA) - Enzymology.658(1): 35–44.doi:10.1016/0005-2744(81)90247-3.PMID6260209.
  9. ^Buchanan, Bob B. (2003-11-28)."Role of Light in the Regulation of Chloroplast Enzymes".Annu. Rev. Plant Physiol.31:341–374.doi:10.1146/annurev.pp.31.060180.002013.
  10. ^abcdHarrison DH, Runquist JA, Holub A, Miziorko HM (April 1998). "The crystal structure of phosphoribulokinase from Rhodobacter sphaeroides reveals a fold similar to that of adenylate kinase".Biochemistry.37(15): 5074–85.doi:10.1021/bi972805y.PMID9548738.
  11. ^abcKono T, Mehrotra S, Endo C, Kizu N, Matusda M, Kimura H, Mizohata E, Inoue T, Hasunuma T, Yokota A, Matsumura H, Ashida H (January 2017)."A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea".Nature Communications.8:14007.Bibcode:2017NatCo...814007K.doi:10.1038/ncomms14007.PMC5241800.PMID28082747.
  12. ^abMiziorko HM, Eckstein F (November 1984)."The stereochemical course of the ribulose-5-phosphate kinase-catalyzed reaction".The Journal of Biological Chemistry.259(21): 13037–40.doi:10.1016/S0021-9258(18)90652-6.PMID6490643.
  13. ^Lebreton S, Gontero B, Avilan L, Ricard J (December 1997)."Information transfer in multienzyme complexes--1. Thermodynamics of conformational constraints and memory effects in the bienzyme glyceraldehyde-3-phosphate-dehydrogenase-phosphoribulokinase complex of Chlamydomonas reinhardtii chloroplasts".European Journal of Biochemistry.250(2): 286–95.doi:10.1111/j.1432-1033.1997.0286a.x.PMID9428675.
  14. ^Wadano A, Nishikawa K, Hirahashi T, Satoh R, Iwaki T (1998-04-01). "Reaction mechanism of phosphoribulokinase from a cyanobacterium, Synechococcus PCC7942".Photosynthesis Research.56(1): 27–33.Bibcode:1998PhoRe..56...27W.doi:10.1023/A:1005979801741.S2CID21409736.
  15. ^abcdHurwitz J (1962).[28c] Phosphoribulokinase.Methods in Enzymology. Vol. 5. pp. 258–261.doi:10.1016/s0076-6879(62)05214-3.ISBN9780121818050.
  16. ^Tabita FR (September 1980)."Pyridine nucleotide control and subunit structure of phosphoribulokinase from photosynthetic bacteria".Journal of Bacteriology.143(3): 1275–80.doi:10.1128/JB.143.3.1275-1280.1980.PMC294495.PMID6251028.
  17. ^Gale NL, Beck JV (September 1966). "Competitive inhibition of phosphoribulokinase by AMP".Biochemical and Biophysical Research Communications.24(5): 792–6.doi:10.1016/0006-291X(66)90396-2.PMID5970515.
  18. ^Ballard RW, MacElroy RD (August 1971). "Phosphoenolpyruvate, a new inhibitor of phosphoribulokinase in pseudomonas facilis".Biochemical and Biophysical Research Communications.44(3): 614–8.doi:10.1016/s0006-291x(71)80127-4.PMID4330777.
  19. ^Gardemann, A.; Stitt, M.; Heldt, H.W. (1983-01-13). "Control of CO2 fixation. Regulation of spinach ribulose-5-phosphate kinase by stromal metabolite levels".Biochimica et Biophysica Acta (BBA) - Bioenergetics.722(1): 51–60.doi:10.1016/0005-2728(83)90156-1.
  20. ^Müller, Bruno (1972-08-01)."A Labile CO2-Fixing Enzyme Complex in Spinach Chloroplasts".Zeitschrift für Naturforschung B.27(8): 925–932.doi:10.1515/znb-1972-0814.
  21. ^Avilan L, Gontero B, Lebreton S, Ricard J (December 1997)."Information transfer in multienzyme complexes--2. The role of Arg64 of Chlamydomonas reinhardtii phosphoribulokinase in the information transfer between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase".European Journal of Biochemistry.250(2): 296–302.doi:10.1111/j.1432-1033.1997.0296a.x.PMID9428676.
  22. ^Nicholson S, Easterby JS, Powls R (January 1987)."Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase".European Journal of Biochemistry.162(2): 423–31.doi:10.1111/j.1432-1033.1987.tb10619.x.PMID3026812.
  23. ^Rault M, Gontero B, Ricard J (May 1991)."Thioredoxin activation of phosphoribulokinase in a chloroplast multi-enzyme complex".European Journal of Biochemistry.197(3): 791–7.doi:10.1111/j.1432-1033.1991.tb15973.x.PMID1851485.
  24. ^Gontero B, Mulliert G, Rault M, Giudici-Orticoni MT, Ricard J (November 1993)."Structural and functional properties of a multi-enzyme complex from spinach chloroplasts. 2. Modulation of the kinetic properties of enzymes in the aggregated state".European Journal of Biochemistry.217(3): 1075–82.doi:10.1111/j.1432-1033.1993.tb18339.x.PMID8223631.
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