Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1992 Apr;98(4):1285–1289. doi: 10.1104/pp.98.4.1285

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Keith R Roesler 1,2,3,1, Beverly L Marcotte 1,2,3, William L Ogren 1,2,3
PMCID: PMC1080346  PMID: 16668789

Abstract

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a Km value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. Km values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The Vmax of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.

Full text

PDF
1289

Images in this article

1286Figure 1
on p.1286
1288Figure 5
on p.1288

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Cotton F. A., Day V. W., Hazen E. E., Jr, Larsen S., Wong S. T. Structure of bis(methylguanidinium) monohydrogen orthophosphate. A model for the arginine-phosphate interactions at the active site of staphylococcal nuclease and other phosphohydrolytic enzymes. J Am Chem Soc. 1974 Jul 10;96(14):4471–4478. doi: 10.1021/ja00821a020. [DOI] [PubMed] [Google Scholar]
  3. Crawford N. A., Sutton C. W., Yee B. C., Johnson T. C., Carlson D. C., Buchanan B. B. Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes. Arch Microbiol. 1984 Oct;139(2-3):124–129. doi: 10.1007/BF00401986. [DOI] [PubMed] [Google Scholar]
  4. Flügge U. I., Stitt M., Freisl M., Heldt H. W. On the Participation of Phosphoribulokinase in the Light Regulation of CO(2) Fixation. Plant Physiol. 1982 Jan;69(1):263–267. doi: 10.1104/pp.69.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gibson J. L., Chen J. H., Tower P. A., Tabita F. R. The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis. Biochemistry. 1990 Sep 4;29(35):8085–8093. doi: 10.1021/bi00487a014. [DOI] [PubMed] [Google Scholar]
  6. Huppe H. C., de Lamotte-Guéry F., Jacquot J-P, Buchanan B. B. The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components. Planta. 1990;180:341–351. [PubMed] [Google Scholar]
  7. Kossmann J., Klintworth R., Bowien B. Sequence analysis of the chromosomal and plasmid genes encoding phosphoribulokinase from Alcaligenes eutrophus. Gene. 1989 Dec 21;85(1):247–252. doi: 10.1016/0378-1119(89)90490-3. [DOI] [PubMed] [Google Scholar]
  8. Meijer W. G., Enequist H. G., Terpstra P., Dijkhuizen L. Nucleotide sequences of the genes encoding fructosebisphosphatase and phosphoribulokinase from Xanthobacter flavus H4-14. J Gen Microbiol. 1990 Nov;136(11):2225–2230. doi: 10.1099/00221287-136-11-2225. [DOI] [PubMed] [Google Scholar]
  9. Milanez S., Mural R. J. Cloning and sequencing of cDNA encoding the mature form of phosphoribulokinase from spinach. Gene. 1988 Jun 15;66(1):55–63. doi: 10.1016/0378-1119(88)90224-7. [DOI] [PubMed] [Google Scholar]
  10. Milanez S., Mural R. J., Hartman F. C. Roles of cysteinyl residues of phosphoribulokinase as examined by site-directed mutagenesis. J Biol Chem. 1991 Jun 5;266(16):10694–10699. [PubMed] [Google Scholar]
  11. Miziorko H. M., Brodt C. A., Krieger T. J. Affinity labeling of spinach leaf phosphoribulokinase by ATP analogs. Modification of an active site lysine. J Biol Chem. 1990 Mar 5;265(7):3642–3647. [PubMed] [Google Scholar]
  12. Moll B., Levine R. P. Characterization of a Photosynthetic Mutant Strain of Chlamydomonas reinhardi Deficient in Phosphoribulokinase Activity. Plant Physiol. 1970 Oct;46(4):576–580. doi: 10.1104/pp.46.4.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Porter M. A., Hartman F. C. Exploration of the function of a regulatory sulfhydryl of phosphoribulokinase from spinach. Arch Biochem Biophys. 1990 Sep;281(2):330–334. doi: 10.1016/0003-9861(90)90452-5. [DOI] [PubMed] [Google Scholar]
  14. Porter M. A., Potter M. D., Hartman F. C. Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16. J Protein Chem. 1990 Aug;9(4):445–451. doi: 10.1007/BF01024620. [DOI] [PubMed] [Google Scholar]
  15. Porter M. A., Stringer C. D., Hartman F. C. Characterization of the regulatory thioredoxin site of phosphoribulokinase. J Biol Chem. 1988 Jan 5;263(1):123–129. [PubMed] [Google Scholar]
  16. RACKER E. The reductive pentose phosphate cycle. I. Phosphoribulokinase and ribulose diphosphate carboxylase. Arch Biochem Biophys. 1957 Jul;69:300–310. doi: 10.1016/0003-9861(57)90496-4. [DOI] [PubMed] [Google Scholar]
  17. Raines C. A., Longstaff M., Lloyd J. C., Dyer T. A. Complete coding sequence of wheat phosphoribulokinase: developmental and light-dependent expression of the mRNA. Mol Gen Genet. 1989 Dec;220(1):43–48. [PubMed] [Google Scholar]
  18. Riordan J. F., McElvany K. D., Borders C. L., Jr Arginyl residues: anion recognition sites in enzymes. Science. 1977 Mar 4;195(4281):884–886. doi: 10.1126/science.190679. [DOI] [PubMed] [Google Scholar]
  19. Roesler K. R., Ogren W. L. Chlamydomonas reinhardtii Phosphoribulokinase : Sequence, Purification, and Kinetics. Plant Physiol. 1990 May;93(1):188–193. doi: 10.1104/pp.93.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Roesler K. R., Ogren W. L. Nucleotide sequence of spinach cDNA encoding phosphoribulokinase. Nucleic Acids Res. 1988 Jul 25;16(14B):7192–7192. doi: 10.1093/nar/16.14.7192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sicher R. C., Jensen R. G. Photosynthesis and ribulose 1,5-bisphosphate levels in intact chloroplasts. Plant Physiol. 1979 Nov;64(5):880–883. doi: 10.1104/pp.64.5.880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Spreitzer R. J., Mets L. Photosynthesis-deficient Mutants of Chlamydomonas reinhardii with Associated Light-sensitive Phenotypes. Plant Physiol. 1981 Mar;67(3):565–569. doi: 10.1104/pp.67.3.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Werdan K., Heldt H. W., Milovancev M. The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark. Biochim Biophys Acta. 1975 Aug 11;396(2):276–292. doi: 10.1016/0005-2728(75)90041-9. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES