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PDBsum entry 1nsm
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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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The catalytic mechanism of galactose mutarotase.
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Authors
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J.B.Thoden,
J.Kim,
F.M.Raushel,
H.M.Holden.
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Ref.
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Protein Sci, 2003,
12,
1051-1059.
[DOI no: ]
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PubMed id
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Abstract
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Galactose mutarotase catalyzes the first step in normal galactose metabolism by
catalyzing the conversion of beta-D-galactose to alpha-D-galactose. The
structure of the enzyme from Lactococcus lactis was recently solved in this
laboratory and shown to be topologically similar to domain 5 of
beta-galactosidase. From this initial X-ray analysis, four amino acid residues
were demonstrated to be intimately involved in sugar binding to the protein: His
96, His 170, Asp 243, and Glu 304. Here we present a combined X-ray
crystallographic and kinetic analysis designed to examine the role of these
residues in the reaction mechanism of the enzyme. For this investigation, the
following site-directed mutant proteins were prepared: H96N, H170N, D243N,
D243A, E304Q, and E304A. All of the structures of these proteins, complexed with
either glucose or galactose, were solved to a nominal resolution of 1.95 A or
better, and their kinetic parameters were measured against D-galactose,
D-glucose, L-arabinose, or D-xylose. From these studies, it can be concluded
that Glu 304 and His 170 are critical for catalysis and that His 96 and Asp 243
are important for proper substrate positioning within the active site.
Specifically, Glu 304 serves as the active site base to initiate the reaction by
removing the proton from the C-1 hydroxyl group of the sugar substrate and His
170 functions as the active site acid to protonate the C-5 ring oxygen.
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Figure 1.
Figure 1. Active site of galactose mutarotase from L.
lactis. A close-up view of the active site within ~5 Å of the
galactose ligand is displayed in (A). Ordered water molecules
surrounding the sugar ligand were omitted for figure clarity.
Both the  - and ß-anomers
of galactose were observed in the electron density map. A
close-up view of the active site within ~5 Å of bound glucose is
presented in (B). All figures in this article were prepared with
the software package MOLSCRIPT (Kraulis 1991).
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Figure 3.
Figure 3. Schematics of hydrogen bonding patterns. Shown in
(A) is the observed hydrogen bonding pattern between the
wild-type protein and galactose. Both the - and ß-anomers
at C-1 are observed in the active site. The hydrogen bonding
pattern exhibited between the E304Q mutant protein and glucose
is depicted in (B).
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The above figures are
reprinted
by permission from the Protein Society:
Protein Sci
(2003,
12,
1051-1059)
copyright 2003.
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