Kinetic mechanism of adenosine 5'-phosphosulphate kinase from rat chondrosarcoma.
Biosynthesis of the activated sulphate donor adenosine 3'-phosphate 5'-phosphosulphate (PAPS) involves the sequential action of two enzyme activities. ATP-sulphurylase catalyses the formation of APS (adenosine 5'-phosphosulphate) from ATP and free sulphate, and APS is then phosphorylated by APS kinase to produce PAPS. Initial-velocity patterns for rat chondrosarcoma APS kinase indicate a single-displacement formal mechanism with KmAPS 76 nM and KmATP = 24 microM. Inhibition studies using analogues of substrates and products were carried out to determine the reaction mechanism. An analogue of PAPS, adenosine 3'-phosphate 5'-[beta-methylene]phosphosulphate, exhibited competitive inhibition with APS and non-competitive inhibition with ATP. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate was also competitive with APS and non-competitive with ATP. Adenosine 5'-[beta gamma-imido]triphosphate showed competitive inhibition with respect to ATP and produced mixed-type inhibition, with a pronounced intercept effect and a small slope effect, with respect to APS. These results are in accord with the formulation of the predominant pathway as a steady-state ordered mechanism with APS as the leading substrate and PAPS as the final product released.
Lyle
S
S
Department of Pediatrics, University of Chicago, IL 60637.
Geller
D H
DH
Ng
K
K
Stanczak
J
J
Westley
J
J
Schwartz
N B
NB
D000250:Adenosine Phosphosulfate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002813:Chondrosarcoma / Q000201:enzymology*; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013430:Sulfate Adenylyltransferase / Q000378:metabolism
D000250:Adenosine Phosphosulfate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D013430:Sulfate Adenylyltransferase; D010744:Phosphoric Monoester Hydrolases; C027342:3'-phosphoadenosine-5'-phosphosulfate phosphohydrolase