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The allosteric enzyme aspartate carbamoyltransferase of Escherichia coli
consists of six regulatory chains (R) and six catalytic chains (C) in D3
symmetry. The less active T conformation, complexed to the allosteric inhibitor
CTP has been refined to 2.6 A (R-factor of 0.155). We now report refinement of
the more active R conformation, complexed to the bisubstrate analog
N-phosphonacetyl-L-aspartate (PALA) to 2.4 A (R-factor of 0.165,
root-mean-square deviations from ideal bond distances and angles of 0.013 A and
2.2 degrees, respectively). The antiparallel beta-sheet in the revised segment
8-65 of the regulatory chain of the T conformation is confirmed in the R
conformation, as is also the interchange of alanine 1 with the side-chain of
asparagine 2 in the catalytic chain. The crystallographic asymmetric unit
containing one-third of the molecule (C2R2) includes 925 sites for water
molecules, and seven side-chains in alternative conformations. The gross
conformational changes of the T to R transition are confirmed, including the
elongation of the molecule along its threefold axis by 12 A, the relative
reorientation of the catalytic trimers C3 by 10 degrees, and the rotation of the
regulatory dimers R2 about the molecular twofold axis by 15 degrees. No changes
occur in secondary structure. Essentially rigid-body transformations account for
the movement of the four domains of each catalytic-regulatory unit; these
include the allosteric effector domain, the equatorial (aspartate) domain, and
the combination of the polar (carbamyl phosphate) and zinc domain, which moves
as a rigid unit. However, interfaces change, for example the interface between
the zinc domain of the R chain and the equatorial domain of the C chain, is
nearly absent in the T state, but becomes extensive in the R state of the
enzyme; also one catalytic-regulatory interface (C1-R4) of the T state
disappears in the more active R state of the enzyme. Segments 50-55, 77-86 and
231-246 of the catalytic chain and segments 51-55, 67-72 and 150-153 of the
regulatory chain show conformational changes that go beyond the rigid-body
movement of their corresponding domains. The localized conformational changes in
the catalytic chain all derive from the interactions of the enzyme with the
inhibitor PALA; these changes may be important for the catalytic mechanism. The
conformation changes in segments 67-72 and 150-153 of the regulatory chain may
be important for the allosteric control of substrate binding. On the basis of
the conformational differences of the T and R states of the enzyme, we present a
plausible scheme for catalysis that assumes the ordered binding of substrates
and the ordered release o
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