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Originally published In Press as doi:10.1074/jbc.M005992200 on August 23, 2000
J. Biol. Chem., Vol. 275, Issue 46, 36303-36310, November 17, 2000
Induction of Positive Cooperativity by Amino Acid Replacements
within the C-terminal Domain of Penicillium chrysogenum
ATP Sulfurylase*
Ian J.
MacRae §,
Eissa
Hanna ¶,
Joseph D.
Ho ¶,
Andrew J.
Fisher , and
Irwin H.
Segel **
From the Section of Molecular and Cellular Biology
and Department of Chemistry, University of California,
Davis, California 95616
ATP sulfurylase from Penicillium
chrysogenum is an allosteric enzyme in which Cys-509 is critical
for maintaining the R state. Cys-509 is located in a C-terminal
domain that is 42% identical to the conserved core of adenosine
5'-phosphosulfate (adenylylsulfate) (APS) kinase. This domain is
believed to provide the binding site for the allosteric effector,
3'-phosphoadenosine 5'-phosphosulfate (PAPS). Replacement of Cys-509
with either Tyr or Ser destabilizes the R state, resulting in an enzyme
that is intrinsically cooperative at pH 8 in the absence of PAPS. The
kinetics of C509Y resemble those of the wild type enzyme in which
Cys-509 has been covalently modified. The kinetics of C509S resemble
those of the wild type enzyme in the presence of PAPS. It is likely
that the negative charge on the Cys-509 side chain helps to stabilize
the R state. Treatment of the enzyme with a low level of trypsin
results in cleavage at Lys-527, a residue that lies in a region
analogous to a PAPS motif-containing mobile loop of true APS kinase.
Both mutant enzymes were cleaved more rapidly than the wild type
enzyme, suggesting that movement of the mobile loop occurs during the R
to T transition.
*
This work was supported in part by National Science
Foundation Grant MCB 9904003 (to I. H. S. and A. J. F.). A
preliminary report was presented at the 18th International Congress of
Biochemistry and Molecular Biology, Birmingham, United Kingdom, July,
2000.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported in part by a Biochemistry and Molecular Biology Training Grant.
¶
Undergraduate honors research students.
**
To whom correspondence should be addressed: Section of Molecular
and Cellular Biology, University of California, One Shields Avenue,
Davis, CA 95616. Tel.: 530-752-3193; Fax: 530-752-3085; E-mail:
ihsegel@ucdavis.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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E. Hanna, K. F. Ng, I. J. MacRae, C. J. Bley, A. J. Fisher, and I. H. Segel
Kinetic and Stability Properties of Penicillium chrysogenum ATP Sulfurylase Missing the C-terminal Regulatory Domain
J. Biol. Chem.,
February 6, 2004;
279(6):
4415 - 4424.
[Abstract]
[Full Text]
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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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