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Literature summary for 3.4.21.111 extracted from

  • Sakaguchi, M.; Osaku, K.; Maejima, S.; Ohno, N.; Sugahara, Y.; Oyama, F.; Kawakita, M.
    Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1 (2014), AMB Express, 4, 59.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.4.21.111

Cloned(Commentary)

Cloned (Comment) Organism
DNAa and amino acid sequence determination and analysis, comparisons of primary structures, expression of wild-type and mutant enzymes fused to the maltose binding protein in Escherichia coli stain TG1, subcloning in Escherichia coli strains DH5alpha and MV1184 Thermus aquaticus

Crystallization (Commentary)

Crystallization (Comment) Organism
crystal structure analysis, overview Thermus aquaticus

Protein Variants

Protein Variants Comment Organism
D138N site-sirected mutagenesis Thermus aquaticus
D17N site-sirected mutagenesis, the mutant is less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis Thermus aquaticus
D183N site-sirected mutagenesis, the disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant results in a significant reduction in thermal stability, and a massive change in the content of the secondary structure compared to the wild-type enzyme Thermus aquaticus
D212N site-sirected mutagenesis, thermal stability of D212N is similar to that of the wild-type enzyme at 70°C, but it is inactivated rapidly at 80°C, the mutant is more prone to unfolding at 80°C than the wild-type enzyme Thermus aquaticus
D58N site-sirected mutagenesis Thermus aquaticus
E237Q site-sirected mutagenesis, the mutant is less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. Disruption of a salt bridge in E237Q results in a rapid decrease of activity during incubation at 70°C and 80°C Thermus aquaticus
G262D site-sirected mutagenesis Thermus aquaticus
G61D site-sirected mutagenesis Thermus aquaticus
additional information amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to the enzyme are replaced to disrupt the bridges one at a time Thermus aquaticus
S277D site-sirected mutagenesis Thermus aquaticus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.74
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D183N Thermus aquaticus
0.77
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D138N Thermus aquaticus
0.79
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, wild-type enzyme Thermus aquaticus
0.8
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant S277D Thermus aquaticus
0.82
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant G61D Thermus aquaticus
0.91
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant E237Q Thermus aquaticus
0.93
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant G262D Thermus aquaticus
0.98
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D17N Thermus aquaticus
0.99
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D58N Thermus aquaticus
1.1
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D212N Thermus aquaticus

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ required Thermus aquaticus

Organism

Organism UniProt Comment Textmining
Thermus aquaticus P08594
-
-
Thermus aquaticus YT-1 P08594
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
-
 Thermus aquaticus N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
-
 ?
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
-
 Thermus aquaticus YT-1 N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
-
 ?

Synonyms

Synonyms Comment Organism
AQN
-
 Thermus aquaticus
aqualysin I
-
 Thermus aquaticus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40
-
 assay at Thermus aquaticus

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
30 90 temperature-dependence of the activity of the wild-type and mutant enzymes, overview Thermus aquaticus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 significance of salt bridges in the thermal stability of the enzyme, overview Thermus aquaticus
70 80 0-120 min, pH 6.0, different stabilities of wild-type and mutant enzymes at 70-80°C, inactivations, overview. The inactivation mechanism of enzyme mutant D212N at 80°C may be different from that of enzyme mutants D17N and E237Q at 70°C Thermus aquaticus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
47.2
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant G61D Thermus aquaticus
53
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D183N Thermus aquaticus
59.5
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D138N Thermus aquaticus
61.3
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant G262D Thermus aquaticus
68.5
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D58N Thermus aquaticus
73.6
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant S277D Thermus aquaticus
75.2
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D212N Thermus aquaticus
91.6
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, wild-type enzyme Thermus aquaticus
96.1
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant E237Q Thermus aquaticus
110
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D17N Thermus aquaticus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Thermus aquaticus

General Information

General Information Comment Organism
evolution the enzyme belongs to the proteinase K subfamily of enzymes Thermus aquaticus
additional information the enzyme has six salt bridges, Arg12-Asp183, Asp17-Arg259, Arg31-Asp237, Arg43-Asp212, Asp58-Arg95 and Asp138-Arg169, in its structure. The common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12 Thermus aquaticus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
63.6
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant G61D Thermus aquaticus
66.2
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant G262D Thermus aquaticus
68.6
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D212N Thermus aquaticus
70
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D58N Thermus aquaticus
71.6
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D183N Thermus aquaticus
77.3
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D138N Thermus aquaticus
92.5
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant S277D Thermus aquaticus
105
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant E237Q Thermus aquaticus
113
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, mutant D17N Thermus aquaticus
116
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, wild-type enzyme Thermus aquaticus