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C68S/S185C
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the ratio of turnover number to Km-value for the substrate L-pyroglutamyl-beta-naphthylamide is 1.3fold higher than the value of the wild-type enzyme. The disulfide bridge between the substituted C185 bonds two subunits, additional 60000 Da band detected by SDS-PAGE. Thermal stability is increased by about 30°C compared to wild-type enzyme
F10Y
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the ratio of turnover number to Km-value is 4.6% of the wild-type value
F13A
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the ratio of turnover number to Km-value is 0.04% of the wild-type value
F13Y
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the ratio of turnover number to Km-value is 51.5% of the wild-type value
F142A
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the ratio of turnover number to Km-value is 0.06% of the wild-type value
F142Y
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the ratio of turnover number to Km-value is 81.3% of the wild-type value
S185C
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the ratio of turnover number to Km-value for the substrate L-pyroglutamyl-beta-naphthylamide is 1.4fold higher than the value of the wild-type enzyme. The disulfide bridge between the substituted C185 bonds two subunits, additional 60000 Da band detected by SDS-PAGE. The mutant enzyme is more stable than the wild-type enzyme at pH 4.0 and at pH 12.0. Thermal stability is increased by about 30°C compared to wild-type enzyme
C210A
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no detectable activity
E101Q
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ca. 2% activity of the native enzyme
E107Q
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ca. 50% activity of the native enzyme
H234S
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no detectable activity
D89N
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35% of activity of the wild-type enzyme
E10Q
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enzyme displays catalytic properties and sensitivities to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide similar to those of wild-type enzyme
E22Q
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enzyme displays catalytic properties and sensitivities to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide similar to wild-type enzyme
A199P
alpha6-helix region of A199P in the D1 state (initial denatured state) is partially unprotected, while some hydrophobic residues are protected against H/D exchange, although these hydrophobic residues are unprotected in the wild-type protein. Structure of A199P in the D1 state forms a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix
C142S
complete loss of the activity
C142S/C188S/E192A
at acidic pH the mutant enzyme is less stable than cysteine-free mutant C142S/C188S. At alkaline pH the mutant enzyme is more stable than cysteine-free mutant C142S/C188S. The thermal stability of the mutant enzyme at pH 2.15, pH 3.04 and pH 7.3 is less than that of the cysteine-free mutant enzyme C142S/C188S. At pH 8.7 and 9.6 the thermal stability of mutant enzyme is higher than that of the cysteine-free mutant C142S/C188S
C142S/C188S/E192D
at acidic pH the mutant enzyme is less stable than cysteine-free mutant C142S/C188S. The thermal stability of the mutant enzyme at pH 2.15, pH 3.04, pH 7.3, pH 8.7 and pH 9.6 is less than that of the cysteine-free mutant enzyme C142S/C188S
C142S/C188S/E192I
at acidic pH the mutant enzyme is less stable than cysteine-free mutant C142S/C188S. At alkaline pH the mutant enzyme is more stable than cysteine-free mutant C142S/C188S. The thermal stability of the mutant enzyme at pH 2.15, pH 3.04 and pH 7.3 is less than that of the cysteine-free mutant enzyme C142S/C188S. At pH 8.7 and 9.6 the thermal stability of mutant enzyme is higher than that of the cysteine-free mutant C142S/C188S
C142S/C188S/E192Q
at acidic pH the mutant enzyme is less stable than cysteine-free mutant C142S/C188S. At alkaline pH the mutant enzyme is more stable than cysteine-free mutant C142S/C188S. The thermal stability of the mutant enzyme at pH 2.15, pH 3.04 and pH 7.3 is less than that of the cysteine-free mutant enzyme C142S/C188S. At pH 8.7 and 9.6 the thermal stability of mutant enzyme is higher than that of the cysteine-free mutant C142S/C188S
C142S/C188S/E192V
at acidic pH the mutant enzyme is less stable than cysteine-free mutant C142S/C188S. At alkaline pH the mutant enzyme is more stable than cysteine-free mutant C142S/C188S. The thermal stability of the mutant enzyme at pH 2.15, pH 3.04 and pH 7.3 is less than that of the cysteine-free mutant enzyme C142S/C188S. At pH 8.7 and 9.6 the thermal stability of mutant enzyme is higher than that of the cysteine-free mutant C142S/C188S
C188S
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activity is reduced by one-fourth relative to the activity of the wild-type enzyme
Cys144Ser/Cys188Ser
cysteine-free variant. The 114-208 segment of the mutant folds into a stable compact structure with non-native helix-helix association in the D1 state. In the folding process from the D1 state to the native state, the alpha4- and alpha6-helices become separated and the central beta-sheet is folded between these helices. The non-native interaction between the alpha4- and alpha6-helices may be responsible for the unusually slow folding of the mutant
additional information
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PPI-deficient mutants show no detectable phenotype, retain infectivity to macrophages in vitro and in mice
C142S/C188S
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the mutant enzyme is used in unfolding and refolding experiments to exclude the complexity in reaction due to oxidation of SH groups
C142S/C188S
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mutant enzyme loses its activity completely
C142S/C188S
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small thermodynamic stability of the mutant enzyme C142S/C188S at low pH. The mutant enzyme is monomeric below pH 2.7, dimeric around pH 3 and tetrameric above pH 4.5. The heat-denaturation of the mutant enzyme is completely reversible at pH 2.3, although the unfolding-refolding reactions are characterized by extremely slow kinetics