Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis.
UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10-30% lower than that of UGPase-2, depending on direction of the reaction, whereas its K(m) values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03-0.14 mM vs. 0.07-0.36 mM, respectively). Both proteins were "true" UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.
Meng
Meng
M
Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, 901 87 Umeå, Sweden.
Wilczynska
Malgorzata
M
Kleczkowski
Leszek A
LA
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase