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PDBsum entry 1alh
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Hydrolase (phosphoric monoester)
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PDB id
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1alh
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Kinetics and crystal structure of a mutant escherichia coli alkaline phosphatase (asp-369--≫asn): a mechanism involving one zinc per active site.
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Authors
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T.T.Tibbitts,
X.Xu,
E.R.Kantrowitz.
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Ref.
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Protein Sci, 1994,
3,
2005-2014.
[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
96%.
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Abstract
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Using site-directed mutagenesis, an aspartate side chain involved in binding
metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369)
was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The
purified mutant enzymes showed reduced turnover rates (kcat) and increased
Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower
than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in
millimolar concentrations to become fully active; even under these conditions
the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of
magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed
that catalysis is 50 times less efficient when the carboxylate side chain of
Asp-369 is replaced by the corresponding amide; and activity is reduced to near
nonenzymic levels when the carboxylate is replaced by a methyl group. The
crystal structure of D369N, solved to 2.5 A resolution with an R-factor of
0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the
kinetic results and the refined X-ray coordinates, a reaction mechanism is
proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1
metal with the possible assistance of a histidine side chain.
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Figure 6.
Fig. 6. Stereo pair showing the electron
density of the anion binding site
ound in D369N alkaline phosphatase.
The ligand at this site, which may be ei-
phosphate or sulfate, was modeled
assulfatedurigthe refinement. Fig-
6,7,and 8 were produced sing the
program SETOR (Evans, 1993).
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Figure 8.
Fig. 8. pair comparing the posi-
tions of side chains, metals, andphos-
phate in the active sites of the D369N
enzyme (thick lines) and the wild-type
enzyme (thin lines). At the I site, zinc
is bound close to he same location in
both enzymes (crosses), but phosphate
(PO,) is more exposed to the surface in
the D369N structure. At the M2 site, zinc
is bound n the wild-type (cross) but not
theutant enzyme. This permits SI02
to move slightly closer to H370 and the
asparagineintroduce at position 369
(D369N). The M3 site normally contains
Mgz+ (cross) with 3 water molecules
(not shown) in the wild-type enzyme; in
the mutant enzyme this space is partially
filled y 1 water molecule (not shown),
the carboxylate side chain of D5 1, and
the#-aminogroup of K328. The 2 sets of
atomiccordinates were aligned using
Quanta to minimize the RMSD of the Cor
atomsbefore making this omparison.
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The above figures are
reprinted
from an Open Access publication published by the Protein Society:
Protein Sci
(1994,
3,
2005-2014)
copyright 1994.
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Secondary reference #1
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Title
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Reaction mechanism of alkaline phosphatase based on crystal structures. Two-Metal ion catalysis.
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Authors
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E.E.Kim,
H.W.Wyckoff.
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Ref.
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J Mol Biol, 1991,
218,
449-464.
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PubMed id
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