title: Purification and characterization of phosphoribulokinase from the marine chromophytic alga Heterosigma carterae.
abstract: In this study we characterized phosphoribulokinase (PRK, EC 2.7.1. 19) from the eukaryotic marine chromophyte Heterosigma carterae. Serial column chromatography resulted in approximately 300-fold purification of the enzyme. A polypeptide of 53 kD was identified as PRK by sequencing the amino terminus of the protein. This protein represents one of the largest composite monomers identified to date for any PRK. The native holoenzyme demonstrated by flow performance liquid chromatography a molecular mass of 214 +/- 12.6 kD, suggesting a tetrameric structure for this catalyst. Because H. carterae PRK activity was insensitive to NADH but was stimulated by dithiothreitol, it appears that the enzyme may require a thioredoxin/ferredoxin rather than a metabolite mode of regulation. Kinetic analysis of this enzyme demonstrated Michaelis constant values of ribulose-5-phosphate (226 microM) and ATP (208 microM), respectively. In summary, H. carterae PRK is unique with respect to holoenzyme structure and function, and thus may represent an alternative evolutionary pathway in Calvin-cycle kinase development.
authors.affiliation: Department of Botany, and School of Oceanography, University of Washington, Seattle, Washington 98195, USA.
authors.forename: T
authors.initials: T
authors.lastname: Hariharan
authors.forename: P J
authors.initials: PJ
authors.lastname: Johnson
authors.forename: R A
authors.initials: RA
authors.lastname: Cattolico
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D056890:Eukaryota / Q000201:enzymology*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
chemical_list: D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase