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PDBsum entry 1jmf
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References listed in PDB file
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Key reference
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Title
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Contributions of orientation and hydrogen bonding to catalysis in asn229 mutants of thymidylate synthase.
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Authors
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J.S.Finer-Moore,
L.Liu,
D.L.Birdsall,
R.Brem,
J.Apfeld,
D.V.Santi,
R.M.Stroud.
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Ref.
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J Mol Biol, 1998,
276,
113-129.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
percentage match of
95%.
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Abstract
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We have determined structures of binary and ternary complexes of five Asn229
variants of thymidylate synthase (TS) and related their structures to the
kinetic constants measured previously. Asn229 forms two hydrogen bonds to the
pyrimidine ring of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). These
hydrogen bonds constrain the orientation of dUMP in binary complexes with dUMP,
and in ternary complexes with dUMP and the TS cofactor,
5,10-methylene-5,6,7,8-tetrahydrofolate. In N229 mutants, where these hydrogen
bonds cannot be made, dUMP binds in a misoriented or more disordered fashion.
Most N229 mutants exhibit no activity for the dehalogenation of 5-bromo-dUMP,
which requires correct orientation of dUMP against Cys198. Since bound dUMP
forms the binding surface against which the pterin ring of cofactor binds,
misorientation of dUMP results in higher Km values for cofactor. At the same
time, binding of the cofactor aids in ordering and positioning dUMP for
catalysis. Hydrophobic mutants, such as N229I, favor an arrangement of solvent
molecules and side-chains around the ligands similar to that in a proposed
transition state for ternary complex formation in wild-type TS, and kcat values
are similar to the wild-type value. Smaller, more hydrophilic mutants favor
arrangements of the solvent and side-chains surrounding the ligands that do not
resemble the proposed transition state. These changes correspond to decreases in
kcat of up to 2000-fold, with only modest increases in Km or Kd. These results
are consistent with the proposal that the hydrogen-bonding network between
water, dUMP and side-chains in the active-site cavity contributes to catalysis
in TS. Asn229 has the unique ability to maintain this critical network, without
sterically interfering with dUMP binding.
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Figure 1.
Figure 1. Proposed mechanism for thymidylate synthase. In
this scheme, AH represents a general base, perhaps the ordered
water molecule Wat1.
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Figure 2.
Figure 2. A drawing of the hydrogen-bond network involving
the pyrimidine ring of dUMP, surrounding residues, and ordered
water molecules in the L. casei TS binary complex with dUMP
[Finer-Moore et al 1993]. Conserved water molecules are labeled
consistently in all drawings.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1998,
276,
113-129)
copyright 1998.
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Secondary reference #1
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Title
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Partitioning roles of side chains in affinity, Orientation, And catalysis with structures for mutant complexes: asparagine-229 in thymidylate synthase.
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Authors
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J.S.Finer-Moore,
L.Liu,
C.E.Schafmeister,
D.L.Birdsall,
T.Mau,
D.V.Santi,
R.M.Stroud.
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Ref.
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Biochemistry, 1996,
35,
5125-5136.
[DOI no: ]
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PubMed id
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Secondary reference #2
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Title
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Stereochemistry of a multistep/bipartite methyl transfer reaction: thymidylate synthase.
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Authors
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R.M.Stroud,
J.S.Finer-Moore.
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Ref.
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Faseb J, 1993,
7,
671-677.
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PubMed id
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Secondary reference #3
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Title
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Refined structures of substrate-Bound and phosphate-Bound thymidylate synthase from lactobacillus casei.
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Authors
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J.Finer-Moore,
E.B.Fauman,
P.G.Foster,
K.M.Perry,
D.V.Santi,
R.M.Stroud.
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Ref.
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J Mol Biol, 1993,
232,
1101-1116.
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PubMed id
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Secondary reference #4
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Title
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Asparagine 229 in thymidylate synthase contributes to, But is not essential for, Catalysis.
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Authors
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L.Liu,
D.V.Santi.
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Ref.
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Proc Natl Acad Sci U S A, 1993,
90,
8604-8608.
[DOI no: ]
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PubMed id
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