Molecular and catalytic properties of Arabidopsis thaliana adenylyl sulfate (APS)-kinase.
A cDNA clone (Atakn1) from Arabidopsis thaliana encoding APS-kinase (EC 2.7.1.25) was investigated for structural and catalytic properties of the gene product. Recombinant his10-AtAkn1 formed PAPS at a Vmax of 7.35 U x mg(-1). The Km for APS was 0.14 microM and for ATP 147 microM. APS caused a severe substrate inhibition (K(i) 4.5 microM). The type of inhibition is uncompetitive with respect to MgATP. High ionic strength and reducing thiols stabilized the enzyme activity. Plant APS-kinase is regulated in vitro by the redox charge with thioredoxin as essential activator. Mutagenesis of a serine in S182C and S182F presumed to be involved in the transfer of the phosphoryl group had no effect upon catalytic activity. Using a yeast two-hybrid system with AtAkn1 as bait, an interacting clone was detected from a cDNA library of A. thaliana cv. Columbia that codes for an APS-kinase iso-form (Atakn2). Complementation of APS-kinase-deficient Saccharomyces cerevisiae met14 showed that AtAkn2 is functionally active as APS-kinase. It was immunologically related to AtAkn1 and presumably represents a plastidal iso-form of the plant APS-kinase gene family.
Lillig
C H
CH
Biochemistry of Plants, Ruhr-University Bochum, Bochum, 44780, Germany.
Schiffmann
S
S
Berndt
C
C
Berken
A
A
Tischka
R
R
Schwenn
J D
JD
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001667:Binding, Competitive; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry; D013997:Time Factors; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012694:Serine; D013879:Thioredoxins; D010088:Oxidoreductases; C052449:3'-phosphoadenylyl-5'-phosphosulfate reductase; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase