APS kinase from Arabidopsis thaliana: genomic organization, expression, and kinetic analysis of the recombinant enzyme.
The gene encoding 5'-adenylylsulfate (APS) kinase (EC 2.7.1.25) (APK) was cloned from Arabidopsis thaliana. There is a single APK locus in A. thaliana. The coding sequence of the gene is composed of 7 exons, interrupted by 6 introns. A transcriptional initiation site was detected 120 bp 5' of the initiation codon. APK mRNA is slightly more abundant in leaves than in roots of A. thaliana and its level does not change in response to sulfur starvation. The APK protein, synthesized in vitro, is able to enter isolated intact chloroplasts. Recombinant APS kinase shows maximal activity at 10 microM APS with 5 mM ATP, but it is inhibited at APS concentrations above 10 microM. The inhibition is alleviated at higher ATP concentrations. Reciprocal plot analysis showed that the theoretical Vmax is approximately 1.2 mumol min-1 mg-1 at 25 degrees C, pH 8.0; the K(m) values are 3.6 microM APS and 1.8 mM ATP.
Lee
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Biotech Center and Plant Science Department, Rutgers University, New Brunswick, New Jersey 08901-8250, USA.
Leustek
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D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005982:Glutathione Transferase / Q000235:genetics; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D010455:Peptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008320:Maltose; D005982:Glutathione Transferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase