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Literature summary for 3.2.1.1 extracted from

  • Nielsen, M.M.; Seo, E.S.; Bozonnet, S.; Aghajari, N.; Robert, X.; Haser, R.; Svensson, B.
    Multi-site substrate binding and interplay in barley alpha-amylase 1 (2008), FEBS Lett., 582, 2567-2571.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
cloned in Escherichia coli DH5alpha and XL10-Gold to pPICZA-AMY1. pPICZA-AMY1 and the mutants Y105A/Y380A, Y105A/Y380M, H395A, Y380A/H395A, Y380A, Y380M, and Y105A are expressed in Pichia pastoris Hordeum vulgare

Crystallization (Commentary)

Crystallization (Comment) Organism
mutants Y105A/Y380A and H395A are crystallized Hordeum vulgare

Protein Variants

Protein Variants Comment Organism
H395A located in the C-domain, the sugar tong, may be involved in the allosteric activation of the enzyme Hordeum vulgare
Y105A 43% of the activity compared to wild-type Hordeum vulgare
Y105A/Y380A loss of 18-36% activity relative to wild-type Hordeum vulgare
Y105A/Y380M loss of 18-36% activity relative to wild-type Hordeum vulgare
Y380A 29-50% decrease in activity compared to wild-type enzyme Hordeum vulgare
Y380A/H395A located in the C-domain, the sugar tong, may be involved in the allosteric activation of the enzyme Hordeum vulgare
Y380M 29-50% decrease in activity compared to wild-type enzyme Hordeum vulgare

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
amylose 0.13 mg/ml, wild-type enzyme, pH 5.5, 30°C, kcat/Km: 1300 1/s/mg/ml Hordeum vulgare
additional information
-
amylose 0.19 mg/ml, H395A mutant, pH 5.5, 30°C, kcat/Km: 394 1/s/mg/ml Hordeum vulgare
additional information
-
amylose 0.30 mg/ml, Y380A/H395A mutant, pH 5.5, 30°C, kcat/Km: 543 1/s/mg/ml Hordeum vulgare
additional information
-
amylose 0.35 mg/ml, Y380M mutant, pH 5.5, 30°C, kcat/Km: 425 1/s/mg/ml Hordeum vulgare
additional information
-
amylose 0.36 mg/ml, Y380A mutant, pH 5.5, 30°C, kcat/Km: 264 1/s/mg/ml Hordeum vulgare
additional information
-
amylose 1.0 mg/ml, Y105A/Y380M mutant, pH 5.5, 30°C, kcat/Km: 56 1/s/mg/ml Hordeum vulgare
additional information
-
amylose 1.2 mg/ml, Y105A/Y380A mutant, pH 5.5, 30°C, kcat/Km: 51 1/s/mg/ml Hordeum vulgare
additional information
-
amylose 1.4 mg/ml, Y105A mutant, pH 5.5, 30°C, kcat/Km: 132 1/s/mg/ml Hordeum vulgare
0.67
-
2-chloro-4-nitrophenyl beta-D-maltoheptaoside Y380A mutant, pH 5.5, 30°C, kcat/Km: 28 1/s/mg/ml Hordeum vulgare
0.7
-
2-chloro-4-nitrophenyl beta-D-maltoheptaoside wild-type enzyme, pH 5.5, 30°C, kcat/Km: 38 1/s/mg/ml Hordeum vulgare
0.87
-
2-chloro-4-nitrophenyl beta-D-maltoheptaoside Y380M mutant, pH 5.5, 30°C, kcat/Km: 39 1/s/mg/ml Hordeum vulgare
0.91
-
2-chloro-4-nitrophenyl beta-D-maltoheptaoside H395A mutant, kcat below 10 1/s, pH 5.5, 30°C Hordeum vulgare
1.4
-
2-chloro-4-nitrophenyl beta-D-maltoheptaoside Y380A/H395A mutant, pH 5.5, 30°C, kcat/Km: 25 1/s/mg/ml Hordeum vulgare
10
-
2-chloro-4-nitrophenyl beta-D-maltoheptaoside Y105A mutant, Km above 10 mM, kcat below 10 1/s, pH 5.5, 30°C Hordeum vulgare
10
-
2-chloro-4-nitrophenyl beta-D-maltoheptaoside Y105A/Y380A mutant, Km above 10 mM, pH 5.5, 30°C, kcat/Km: 1300 1/s/mg/ml Hordeum vulgare
10
-
2-chloro-4-nitrophenyl beta-D-maltoheptaoside Y105A/Y380M mutant, Km above 10 mM, kcat below 10 1/s, pH 5.5, 30°C Hordeum vulgare

Organism

Organism UniProt Comment Textmining
Hordeum vulgare P00693
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
substrate insoluble Blue Starch, pH 6.8, 37°C, wild-type: 2800 U/mg, mutants Y105A/Y380A: 2300 U/ml, Y105A/Y380M: 1800 U/ml, H395A: 2300 U/ml, Y380A/H395A: 3200 U/ml, Y380A: 1400 U/ml, Y380M: 2000 U/ml, and Y105A: 4000 U/ml. Units defined as 1 Unit gives DELTA optical density at 620 nm: 1. Y105A/Y380A and Y105A/Y380M mutant lose 18-36% activity relative to wild-type. 29-50% decrease in acitivity of the single mutants Y380M and Y380A. Hordeum vulgare

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside + H2O pH 6.8, 30°C Hordeum vulgare 2-chloro-4-nitrophenol + ?
-
?
amylose + H2O amylose DP440, pH 6.8, 30°C Hordeum vulgare ?
-
?
insoluble Blue Starch + H2O pH 5.5, 37°C Hordeum vulgare ?
-
?

Synonyms

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

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at, Michaelis-Menten kinetics and kcat determination Hordeum vulgare
37
-
standard assay at Hordeum vulgare

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3 6 2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside wild-type enzyme, pH 5.5, 30°C, kcat/Km: 48 1/s/mg/ml Hordeum vulgare
10
-
2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside Y105A mutant, kcat below 10 1/s, pH 5.5, 30°C Hordeum vulgare
10
-
2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside Y105A/Y380A mutant, kcat below 10 1/s, pH 5.5, 30°C Hordeum vulgare
10
-
2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside Y105A/Y380M mutant, kcat below 10 1/s, pH 5.5, 30°C Hordeum vulgare
19
-
2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside Y380A mutant, pH 5.5, 30°C, kcat/Km: 28 1/s/mg/ml Hordeum vulgare
21
-
2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside H395A mutant, pH 5.5, 30°C, kcat/Km: 23 1/s/mg/ml Hordeum vulgare
34
-
2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside Y380M mutant, pH 5.5, 30°C, kcat/Km: 39 1/s/mg/ml Hordeum vulgare
35
-
2-chloro-4-nitrophenyl beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-4)-beta-D-glucopyranoside Y380A/H395A mutant, pH 5.5, 30°C, kcat/Km: 25 1/s/mg/ml Hordeum vulgare
56
-
amylose Y105A/Y380M mutant, pH 5.5, 30°C, kcat/Km: 56 1/s/mg/ml, kcat/Km decreases 10-25fold for all Y105 mutants Hordeum vulgare
61
-
amylose Y105A/Y380A mutant, pH 5.5, 30°C, kcat/Km: 51 1/s/mg/ml, kcat/Km decreases 10-25fold for all Y105 mutants Hordeum vulgare
75
-
amylose H395A mutant, pH 5.5, 30°C, kcat/Km: 394 1/s/mg/ml Hordeum vulgare
95
-
amylose Y380A mutant, pH 5.5, 30°C, kcat/Km: 264 1/s/mg/ml Hordeum vulgare
149
-
amylose Y380M mutant, pH 5.5, 30°C, kcat/Km: 425 1/s/mg/ml Hordeum vulgare
163
-
amylose Y380A/H395A mutant, pH 5.5, 30°C, kcat/Km: 543 1/s/mg/ml Hordeum vulgare
169
-
amylose wild-type enzyme, pH 5.5, 30°C, kcat/Km: 1300 1/s/mg/ml Hordeum vulgare
178
-
amylose Y105A mutant, pH 5.5, 30°C, kcat/Km: 132 1/s/mg/ml, kcat/Km decreases 10-25fold for all Y105 mutants Hordeum vulgare

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.5
-
standard assay at Hordeum vulgare
6.8
-
assay at, Michaelis-Menten kinetics and kcat determination Hordeum vulgare