Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma.
ATP-sulphurylase catalyses the production of adenosine 5'-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3'-phosphate 5'-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction.
Lyle
S
S
Department of Pediatrics, University of Chicago, IL 60637.
Geller
D H
DH
Ng
K
K
Westley
J
J
Schwartz
N B
NB
D000250:Adenosine Phosphosulfate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002704:Chlorates / Q000494:pharmacology; D002813:Chondrosarcoma / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D013430:Sulfate Adenylyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism
D002704:Chlorates; D010710:Phosphates; D013431:Sulfates; D000250:Adenosine Phosphosulfate; D000255:Adenosine Triphosphate; D013430:Sulfate Adenylyltransferase; D008274:Magnesium