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PDBsum entry 1kgu
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Contents |
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* Residue conservation analysis
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PDB id:
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Hydrolase
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Title:
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Three dimensional structure analysis of the r337a variant of human pancreatic alpha-amylase
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Structure:
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Alpha-amylase, pancreatic. Chain: a. Synonym: 1,4-alpha-d-glucan glucanohydrolase, pancreatic alpha- amylase, pa. Engineered: yes. Mutation: yes
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Source:
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Homo sapiens. Human. Organism_taxid: 9606. Expressed in: pichia pastoris. Expression_system_taxid: 4922
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Resolution:
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Authors:
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S.Numao,R.Maurus,G.Sidhu,Y.Wang,C.M.Overall,G.D.Brayer,S.G.Withers
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Key ref:
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S.Numao
et al.
(2002).
Probing the role of the chloride ion in the mechanism of human pancreatic alpha-amylase.
Biochemistry,
41,
215-225.
PubMed id:
DOI:
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Date:
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28-Nov-01
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Release date:
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16-Jan-02
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PROCHECK
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Headers
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References
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P04746
(AMYP_HUMAN) -
Pancreatic alpha-amylase from Homo sapiens
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Seq:
Struc:
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511 a.a.
496 a.a.*
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Key: |
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PfamA domain |
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Secondary structure |
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CATH domain |
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*
PDB and UniProt seqs differ
at 2 residue positions (black
crosses)
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Enzyme class:
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E.C.3.2.1.1
- alpha-amylase.
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Reaction:
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Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.
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DOI no:
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Biochemistry
41:215-225
(2002)
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PubMed id:
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Probing the role of the chloride ion in the mechanism of human pancreatic alpha-amylase.
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S.Numao,
R.Maurus,
G.Sidhu,
Y.Wang,
C.M.Overall,
G.D.Brayer,
S.G.Withers.
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ABSTRACT
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Human pancreatic alpha-amylase (HPA) is a member of the alpha-amylase family
involved in the degradation of starch. Some members of this family, including
HPA, require chloride for maximal activity. To determine the mechanism of
chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q)
were made in which residues in the chloride ion binding site were replaced.
Mutations in this binding site were found to severely affect the ability of HPA
to bind chloride ions with no binding detected for the R195 and R337 mutant
enzymes. X-ray crystallographic analysis revealed that these mutations did not
result in significant structural changes. However, the introduction of these
mutations did alter the kinetic properties of the enzyme. Mutations to residue
R195 resulted in a 20-450-fold decrease in the activity of the enzyme toward
starch and shifted the pH optimum to a more basic pH. Interestingly, replacement
of R337 with a nonbasic amino acid resulted in an alpha-amylase that no longer
required chloride for catalysis and has a pH profile similar to that of
wild-type HPA. In contrast, a mutation at residue N298 resulted in an enzyme
that had much lower binding affinity for chloride but still required chloride
for maximal activity. We propose that the chloride is required to increase the
pK(a) of the acid/base catalyst, E233, which would otherwise be lower due to the
presence of R337, a positively charged residue.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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F.Cardona,
A.Goti,
C.Parmeggiani,
P.Parenti,
M.Forcella,
P.Fusi,
L.Cipolla,
S.M.Roberts,
G.J.Davies,
and
T.M.Gloster
(2010).
Casuarine-6-O-alpha-D-glucoside and its analogues are tight binding inhibitors of insect and bacterial trehalases.
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Chem Commun (Camb),
46,
2629-2631.
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PDB code:
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J.Pytelková,
J.Hubert,
M.Lepsík,
J.Sobotník,
R.Sindelka,
I.Krízková,
M.Horn,
and
M.Mares
(2009).
Digestive alpha-amylases of the flour moth Ephestia kuehniella--adaptation to alkaline environment and plant inhibitors.
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FEBS J,
276,
3531-3546.
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C.A.Tarling,
K.Woods,
R.Zhang,
H.C.Brastianos,
G.D.Brayer,
R.J.Andersen,
and
S.G.Withers
(2008).
The search for novel human pancreatic alpha-amylase inhibitors: high-throughput screening of terrestrial and marine natural product extracts.
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Chembiochem,
9,
433-438.
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J.C.Marx,
J.Poncin,
J.P.Simorre,
P.W.Ramteke,
and
G.Feller
(2008).
The noncatalytic triad of alpha-amylases: a novel structural motif involved in conformational stability.
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Proteins,
70,
320-328.
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S.Cheluvaraja,
M.Mihailescu,
and
H.Meirovitch
(2008).
Entropy and free energy of a mobile protein loop in explicit water.
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J Phys Chem B,
112,
9512-9522.
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A.J.Plested,
and
M.L.Mayer
(2007).
Structure and mechanism of kainate receptor modulation by anions.
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Neuron,
53,
829-841.
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PDB code:
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R.Maurus,
A.Begum,
H.H.Kuo,
A.Racaza,
S.Numao,
C.Andersen,
J.W.Tams,
J.Vind,
C.M.Overall,
S.G.Withers,
and
G.D.Brayer
(2005).
Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase.
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Protein Sci,
14,
743-755.
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PDB codes:
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The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
Where a reference describes a PDB structure, the PDB
code is
shown on the right.
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Links
