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.
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Research Article|
July 15 1994
Kinetic mechanism of adenosine 5′-phosphosulphate kinase from rat chondrosarcoma
S Lyle;
S Lyle
1Departments of Pediatrics and Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, U.S.A.
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D H Geller;
D H Geller
1Departments of Pediatrics and Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, U.S.A.
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K Ng;
K Ng
1Departments of Pediatrics and Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, U.S.A.
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J Stanczak;
J Stanczak
1Departments of Pediatrics and Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, U.S.A.
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J Westley;
J Westley
1Departments of Pediatrics and Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, U.S.A.
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N B Schwartz
N B Schwartz
1Departments of Pediatrics and Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, U.S.A.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1994 The Biochemical Society, London
1994
Biochem J (1994) 301 (2): 355–359.
Citation
S Lyle, D H Geller, K Ng, J Stanczak, J Westley, N B Schwartz; Kinetic mechanism of adenosine 5′-phosphosulphate kinase from rat chondrosarcoma. Biochem J 15 July 1994; 301 (2): 355–359. doi: https://doi.org/10.1042/bj3010355
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