Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction.
Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor. APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS. Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction. Similar pre-steady-state kinetic measurements show that the rate constant for transfer of the phosphoryl group from E-P to APS is 91 s-1. Thus, the phosphorylated enzyme is kinetically competent to be on the reaction path. In order to elucidate which amino acid residue is phosphorylated, and thus to define the active site region of APS kinase, we have determined the complete sequence of cysC, the structural gene for this enzyme in E. coli. The coding region contains 603 nucleotides and encodes a protein of 22,321 Da. Near the amino terminus is the sequence 35GLSGSGKS, which exemplifies a motif known to interact with the beta-phosphoryl group of purine nucleotides. The residue that is phosphorylated upon incubation with ATP has been identified as serine-109 on the basis of the amino acid composition of a radiolabeled peptide purified from a proteolytic digest of 32P-labeled enzyme. We have identified a sequence beginning at residue 147 which may reflect a PAPS binding site. This sequence was identified in the carboxy terminal region of 10 reported sequences of proteins of PAPS metabolism.
Satishchandran
C
C
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Hickman
Y N
YN
Markham
G D
GD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005796:Genes; D007700:Kinetics; D008969:Molecular Sequence Data; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D017385:Sequence Homology
D010768:Phosphoserine; D010724:Phosphoadenosine Phosphosulfate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase