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 (Comment) | Organism |
---|---|
mutants Y105A/Y380A and H395A are crystallized | Hordeum vulgare |
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 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 | UniProt | Comment | Textmining |
---|---|---|---|
Hordeum vulgare | P00693 | - |
- |
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 | 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 | Comment | Organism |
---|---|---|
AMY1 | - |
Hordeum vulgare |
barley alpha-amylase 1 | - |
Hordeum vulgare |
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 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 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 |