Domain Bridging Interactions
A NECESSARY CONTRIBUTION TO THE FUNCTION AND STRUCTURE OF
ESCHERICHIA COLI ASPARTATE TRANSCARBAMOYLASE*
Jessica B.
Sakash
,
Mark K.
Williams,
Hiro
Tsuruta§, and
Evan
R.
Kantrowitz¶
From the Department of Chemistry, Boston College,
Merkert Chemistry Center, Chestnut Hill, Massachusetts 02467 and the
§ Stanford Synchrotron Radiation Laboratory, SLAC,
Stanford, California 94309-0210
Aspartate transcarbamoylase undergoes a domain
closure in the catalytic chains upon binding of the substrates that
initiates the allosteric transition. Interdomain bridging interactions
between Glu50 and both Arg167 and
Arg234 have been shown to be critical for stabilization of
the R state. A hybrid version of the enzyme has been generated in
vitro containing one wild-type catalytic subunit, one catalytic
subunit in which Glu50 in each catalytic chain has been
replaced by Ala (E50A), and wild-type regulatory subunits. Thus,
the hybrid enzyme has one catalytic subunit capable of domain
closure and one catalytic subunit incapable of domain closure. The
hybrid does not behave as a simple mixture of the constituent subunits;
it exhibits lower catalytic activity and higher aspartate affinity than
would be expected. As opposed to the wild-type enzyme, the hybrid is
inhibited allosterically by CTP at saturating substrate concentrations. As opposed to the E50A holoenzyme, the hybrid is not allosterically activated by ATP at saturating substrate concentrations. Small angle
x-ray scattering showed that three of the six interdomain bridging
interactions in the hybrid is sufficient to cause the global structural
change to the R state, establishing the critical nature of these
interactions for the allosteric transition of aspartate transcarbamoylase.
*
This work was supported by Grant GM26237 from the National
Institutes of Health. The Stanford Synchrotron Radiation Laboratory is
operated by the Department of Energy, Office of Basic Energy Sciences.
The Stanford Synchrotron Radiation Laboratory Structural Biology
Resource is supported by the National Institutes of Health, by National
Center for Research Resources Grant P41RR01209, and by the Department
of Energy, Office of Biological and Environmental Research.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.
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