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Literature summary for 3.2.1.1 extracted from

  • Bak-Jensen, K.S.; Andre, G.; Gottschalk, T.E.; Paes, G.; Tran, V.; Svensson, B.
    Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1 (2004), J. Biol. Chem., 279, 10093-10102.
    View publication on PubMed
Search for more literature for 3.2.1.1

Cloned(Commentary)

Cloned (Comment) Organism
AMY1, DNA and amino acid sequence determination and analysis, cloning in Escherichia coli strain DH5alpha, expression of wild-type and mutant enzymes in Pichia pastoris strain GS115 Hordeum vulgare

Protein Variants

Protein Variants Comment Organism
T212P site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme Hordeum vulgare
T212W site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme Hordeum vulgare
T212Y site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme Hordeum vulgare
Y105A site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme Hordeum vulgare
Y105A/T212W site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme Hordeum vulgare
Y105A/T212Y site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme Hordeum vulgare
Y105F site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme Hordeum vulgare
Y105W site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme Hordeum vulgare

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.12
-
 amylose DP17 pH 5.5, 37°C, mutant T212Y Hordeum vulgare
0.29
-
 amylose DP17 pH 5.5, 37°C, mutant T212W Hordeum vulgare
0.3
-
 insoluble blue starch pH 5.5, 37°C, mutant T212W Hordeum vulgare
0.3
-
 insoluble blue starch pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
0.4
-
 insoluble blue starch below, pH 5.5, 37°C, mutant Y105A Hordeum vulgare
0.4
-
 insoluble blue starch pH 5.5, 37°C, mutant Y105F Hordeum vulgare
0.4
-
 insoluble blue starch pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
0.48
-
 amylose DP17 pH 5.5, 37°C, mutants Y105W and T212P Hordeum vulgare
0.5
-
 insoluble blue starch pH 5.5, 37°C, mutant Y105W Hordeum vulgare
0.57
-
 amylose DP17 pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
0.58
-
 amylose DP17 pH 5.5, 37°C, mutant Y105F Hordeum vulgare
0.6
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212W Hordeum vulgare
0.6
-
 insoluble blue starch pH 5.5, 37°C, mutant T212Y Hordeum vulgare
1
-
 insoluble blue starch pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
1.3
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105W Hordeum vulgare
1.4
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212P Hordeum vulgare
1.5
-
 insoluble blue starch pH 5.5, 37°C, mutant T212P Hordeum vulgare
1.7
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
2
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212Y Hordeum vulgare
2
-
 amylose DP17 pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
2.27
-
 amylose DP17 pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
2.36
-
 amylose DP17 below, pH 5.5, 37°C, mutant Y105A Hordeum vulgare
3.1
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
4.9
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105F Hordeum vulgare
6
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
10
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside above, pH 5.5, 37°C, mutant Y105A Hordeum vulgare

Organism

Organism UniProt Comment Textmining
Hordeum vulgare P04063
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Pichia pastoris strain GS115 by beta-cyclodextrin affinity chromatography Hordeum vulgare

Reaction

Reaction Comment Organism Reaction ID
(alpha-D-glucopyranosyl-(1-4))n-alpha-D-glucopyranose + H2O = (alpha-D-glucopyranosyl-(1-4))n-m-alpha-D-glucopyranose + (alpha-D-glucopyranosyl-(1-4))m-alpha-D-glucopyranose Tyr105 and Thr212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of alpha-amylase 1 Hordeum vulgare
a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
-
 Hordeum vulgare

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-chloro-4-nitrophenyl beta-D-maltoheptaoside + H2O
-
 Hordeum vulgare 2-chloro-4-nitrophenol + beta-D-maltoheptaose
-
 ?
amylose + H2O amylose DP17, preferred substrate of mutant enzyme T212Y Hordeum vulgare oligosaccharides
-
 ?
amylose DP17 + H2O
-
 Hordeum vulgare ?
-
 ?
additional information substrate specificities of wild-type and mutant enzymes, oligosaccharide action patterns of mutant enzymes, overview, Tyr105 and Thr212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of alpha-amylase 1, modelling of oligosaccharide substrate docking at the substrate binding area Hordeum vulgare ?
-
 ?
starch + H2O insoluble blue starch, preferred substrate of mutant enzyme Y105A Hordeum vulgare oligosaccharides
-
 ?

Synonyms

Synonyms Comment Organism
alpha-amylase 1
-
 Hordeum vulgare
AMY1
-
 Hordeum vulgare

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Hordeum vulgare

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
10
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside below, pH 5.5, 37°C, mutant Y105A Hordeum vulgare
24
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
31
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
61
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212W Hordeum vulgare
78
-
 amylose DP17 pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
105
-
 amylose DP17 pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
119
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
127
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212Y Hordeum vulgare
127
-
 amylose DP17 pH 5.5, 37°C, mutant T212Y Hordeum vulgare
132
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105W Hordeum vulgare
140
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212P Hordeum vulgare
146
-
 amylose DP17 below, pH 5.5, 37°C, mutant Y105A Hordeum vulgare
154
-
 amylose DP17 pH 5.5, 37°C, mutant T212W Hordeum vulgare
158
-
 amylose DP17 pH 5.5, 37°C, mutant Y105F Hordeum vulgare
165
-
 amylose DP17 pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
200
-
 amylose DP17 pH 5.5, 37°C, mutant Y105W Hordeum vulgare
203
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105F Hordeum vulgare
264
-
 amylose DP17 pH 5.5, 37°C, mutant T212P Hordeum vulgare

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.5
-
 assay at Hordeum vulgare