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Literature summary 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

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.1 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

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.1 0.12
-
 amylose DP17 pH 5.5, 37°C, mutant T212Y Hordeum vulgare
3.2.1.1 0.29
-
 amylose DP17 pH 5.5, 37°C, mutant T212W Hordeum vulgare
3.2.1.1 0.3
-
 insoluble blue starch pH 5.5, 37°C, mutant T212W Hordeum vulgare
3.2.1.1 0.3
-
 insoluble blue starch pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
3.2.1.1 0.4
-
 insoluble blue starch below, pH 5.5, 37°C, mutant Y105A Hordeum vulgare
3.2.1.1 0.4
-
 insoluble blue starch pH 5.5, 37°C, mutant Y105F Hordeum vulgare
3.2.1.1 0.4
-
 insoluble blue starch pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
3.2.1.1 0.48
-
 amylose DP17 pH 5.5, 37°C, mutants Y105W and T212P Hordeum vulgare
3.2.1.1 0.5
-
 insoluble blue starch pH 5.5, 37°C, mutant Y105W Hordeum vulgare
3.2.1.1 0.57
-
 amylose DP17 pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
3.2.1.1 0.58
-
 amylose DP17 pH 5.5, 37°C, mutant Y105F Hordeum vulgare
3.2.1.1 0.6
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212W Hordeum vulgare
3.2.1.1 0.6
-
 insoluble blue starch pH 5.5, 37°C, mutant T212Y Hordeum vulgare
3.2.1.1 1
-
 insoluble blue starch pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
3.2.1.1 1.3
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105W Hordeum vulgare
3.2.1.1 1.4
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212P Hordeum vulgare
3.2.1.1 1.5
-
 insoluble blue starch pH 5.5, 37°C, mutant T212P Hordeum vulgare
3.2.1.1 1.7
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
3.2.1.1 2
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212Y Hordeum vulgare
3.2.1.1 2
-
 amylose DP17 pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
3.2.1.1 2.27
-
 amylose DP17 pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
3.2.1.1 2.36
-
 amylose DP17 below, pH 5.5, 37°C, mutant Y105A Hordeum vulgare
3.2.1.1 3.1
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
3.2.1.1 4.9
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105F Hordeum vulgare
3.2.1.1 6
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
3.2.1.1 10
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside above, pH 5.5, 37°C, mutant Y105A Hordeum vulgare

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.1 Hordeum vulgare P04063
-
-

Purification (Commentary)

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

Reaction

EC Number Reaction Comment Organism Reaction ID
3.2.1.1 (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]

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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.1 2-chloro-4-nitrophenyl beta-D-maltoheptaoside + H2O
-
 Hordeum vulgare 2-chloro-4-nitrophenol + beta-D-maltoheptaose
-
 ?
3.2.1.1 amylose + H2O amylose DP17, preferred substrate of mutant enzyme T212Y Hordeum vulgare oligosaccharides
-
 ?
3.2.1.1 amylose DP17 + H2O
-
 Hordeum vulgare ?
-
 ?
3.2.1.1 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 ?
-
 ?
3.2.1.1 starch + H2O insoluble blue starch, preferred substrate of mutant enzyme Y105A Hordeum vulgare oligosaccharides
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.2.1.1 alpha-amylase 1
-
 Hordeum vulgare
3.2.1.1 AMY1
-
 Hordeum vulgare

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.1 37
-
 assay at Hordeum vulgare

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.1 10
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside below, pH 5.5, 37°C, mutant Y105A Hordeum vulgare
3.2.1.1 24
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
3.2.1.1 31
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
3.2.1.1 61
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212W Hordeum vulgare
3.2.1.1 78
-
 amylose DP17 pH 5.5, 37°C, mutant Y105A/T212Y Hordeum vulgare
3.2.1.1 105
-
 amylose DP17 pH 5.5, 37°C, mutant Y105A/T212W Hordeum vulgare
3.2.1.1 119
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
3.2.1.1 127
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212Y Hordeum vulgare
3.2.1.1 127
-
 amylose DP17 pH 5.5, 37°C, mutant T212Y Hordeum vulgare
3.2.1.1 132
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105W Hordeum vulgare
3.2.1.1 140
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant T212P Hordeum vulgare
3.2.1.1 146
-
 amylose DP17 below, pH 5.5, 37°C, mutant Y105A Hordeum vulgare
3.2.1.1 154
-
 amylose DP17 pH 5.5, 37°C, mutant T212W Hordeum vulgare
3.2.1.1 158
-
 amylose DP17 pH 5.5, 37°C, mutant Y105F Hordeum vulgare
3.2.1.1 165
-
 amylose DP17 pH 5.5, 37°C, wild-type enzyme Hordeum vulgare
3.2.1.1 200
-
 amylose DP17 pH 5.5, 37°C, mutant Y105W Hordeum vulgare
3.2.1.1 203
-
 2-chloro-4-nitrophenyl beta-D-maltoheptaoside pH 5.5, 37°C, mutant Y105F Hordeum vulgare
3.2.1.1 264
-
 amylose DP17 pH 5.5, 37°C, mutant T212P Hordeum vulgare

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.1 5.5
-
 assay at Hordeum vulgare