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PDBsum entry 2nrc
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References listed in PDB file
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Key reference
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Title
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Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-Coa:3-Ketoacid coenzyme a transferase on binding coenzyme a.
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Authors
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S.D.Tammam,
J.C.Rochet,
M.E.Fraser.
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Ref.
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Biochemistry, 2007,
46,
10852-10863.
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PubMed id
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Abstract
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Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA
to acetoacetate via a thioester intermediate with its active site glutamate
residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now
be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), reflecting a
conformational change of SCOT upon formation of the thioester. Since either Cys
28 or Cys 196 could be the target, each was mutated to Ser to distinguish
between them. Like wild-type SCOT, the C196S mutant protein was modified rapidly
in the presence of acyl-CoA substrates. In contrast, the C28S mutant protein was
modified much more slowly under identical conditions, indicating that Cys 28 is
the residue exposed on binding CoA. The specific activity of the C28S mutant
protein was unexpectedly lower than that of wild-type SCOT. X-ray
crystallography revealed that Ser adopts a different conformation than the
native Cys. A chloride ion is bound to one of four active sites in the crystal
structure of the C28S mutant protein, mimicking substrate, interacting with Lys
329, Asn 51, and Asn 52. On the basis of these results and the studies of the
structurally similar CoA transferase from Escherichia coli, YdiF, bound to CoA,
the conformational change in SCOT was deduced to be a domain rotation of 17
degrees coupled with movement of two loops: residues 321-329 that bury Cys 28
and interact with succinate or acetoacetate and residues 374-386 that interact
with CoA. Modeling this conformational change has led to the proposal of a new
mechanism for catalysis by SCOT.
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