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PDBsum entry 1kbb
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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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Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids
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Structure:
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Alpha-amylase, pancreatic. Chain: a. Synonym: 1,4-alpha-d-glucan glucanohydrolase, pancreatic alpha- amylase. 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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1.90Å
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R-factor:
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0.172
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R-free:
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0.194
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Authors:
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E.H.Rydberg,C.Li,R.Maurus,C.M.Overall,G.D.Brayer,S.G.Withers
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Key ref:
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E.H.Rydberg
et al.
(2002).
Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.
Biochemistry,
41,
4492-4502.
PubMed id:
DOI:
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Date:
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05-Nov-01
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Release date:
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10-Apr-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
41:4492-4502
(2002)
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PubMed id:
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Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.
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E.H.Rydberg,
C.Li,
R.Maurus,
C.M.Overall,
G.D.Brayer,
S.G.Withers.
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ABSTRACT
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The roles of three conserved active site carboxylic acids (D197, E233, and D300)
in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied
by utilizing site-directed mutagenesis in combination with structural and
kinetic analyses of the resultant enzymes. All three residues were mutated to
both alanine and the respective amide, and a double alanine mutant (E233A/D300A)
was also generated. Structural analyses demonstrated that there were no
significant differences in global fold for the mutant enzymes. Kinetic analyses
were performed on the mutants, utilizing a range of substrates. All results
suggested that D197 was the nucleophile, as virtually all activity
(>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at
this position when assayed with several substrates. The significantly greater
second-order rate constant of E233 mutants on "activated" substrates
(k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1))
compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose =
0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid
catalyst, as did the pH-activity profiles. Transglycosylation was favored over
hydrolysis for the reactions of several of the enzymes mutated at D300. At the
least, this suggests an overall impairment of the catalytic mechanism where the
reaction then proceeds using the better acceptor (oligosaccharide instead of
water). This may also suggest that D300 plays a crucial role in enzymic
interactions with the nucleophilic water during the hydrolysis of the glycosidic
bond.
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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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L.M.Willis,
R.Zhang,
A.Reid,
S.G.Withers,
and
W.W.Wakarchuk
(2009).
Mechanistic investigation of the endo-alpha-N-acetylgalactosaminidase from Streptococcus pneumoniae R6.
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Biochemistry,
48,
10334-10341.
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B.Cobucci-Ponzano,
F.Conte,
M.Rossi,
and
M.Moracci
(2008).
The alpha-L: -fucosidase from Sulfolobus solfataricus.
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Extremophiles,
12,
61-68.
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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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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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R.Quezada-Calvillo,
C.C.Robayo-Torres,
Z.Ao,
B.R.Hamaker,
A.Quaroni,
G.D.Brayer,
E.E.Sterchi,
S.S.Baker,
and
B.L.Nichols
(2007).
Luminal substrate "brake" on mucosal maltase-glucoamylase activity regulates total rate of starch digestion to glucose.
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J Pediatr Gastroenterol Nutr,
45,
32-43.
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S.Z.Fisher,
L.Govindasamy,
C.Tu,
M.Agbandje-McKenna,
D.N.Silverman,
H.J.Rajaniemi,
and
R.McKenna
(2006).
Structure of human salivary alpha-amylase crystallized in a C-centered monoclinic space group.
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Acta Crystallogr Sect F Struct Biol Cryst Commun,
62,
88-93.
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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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N.Ramasubbu,
C.Ragunath,
P.J.Mishra,
L.M.Thomas,
G.Gyémánt,
and
L.Kandra
(2004).
Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.
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Eur J Biochem,
271,
2517-2529.
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PDB codes:
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S.Numao,
I.Damager,
C.Li,
T.M.Wrodnigg,
A.Begum,
C.M.Overall,
G.D.Brayer,
and
S.G.Withers
(2004).
In situ extension as an approach for identifying novel alpha-amylase inhibitors.
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J Biol Chem,
279,
48282-48291.
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PDB codes:
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C.A.Tarling,
S.He,
G.Sulzenbacher,
C.Bignon,
Y.Bourne,
B.Henrissat,
and
S.G.Withers
(2003).
Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis.
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J Biol Chem,
278,
47394-47399.
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J.E.Nielsen,
and
J.A.McCammon
(2003).
Calculating pKa values in enzyme active sites.
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Protein Sci,
12,
1894-1901.
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Y.Tatara,
B.R.Lee,
T.Yoshida,
K.Takahashi,
and
E.Ichishima
(2003).
Identification of catalytic residues of Ca2+-independent 1,2-alpha-D-mannosidase from Aspergillus saitoi by site-directed mutagenesis.
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J Biol Chem,
278,
25289-25294.
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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
codes are
shown on the right.
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Links
