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 μM APS with 5 mM ATP, but it is inhibited at APS concentrations above 10 μM. The inhibition is alleviated at higher ATP concentrations. Reciprocal plot analysis showed that the theoretical V(max) is ~ 1.2 μmol min^-1 mg^-1 at 25°C, pH 8.0; the K(m) values are 3.6 μM APS and 1.8 mM ATP.