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3.4.24.56: insulysin

This is an abbreviated version!
For detailed information about insulysin, go to the full flat file.

Word Map on EC 3.4.24.56

Reaction

Degradation of insulin, glucagon and other polypeptides. No action on proteins =

Synonyms

ADE, amyloid degrading enzyme, cgd6_5510, EC 3.4.22.11, EC 3.4.99.10, EC 3.4.99.45, gamma-endorphin-generating enzyme, IDE, INS20-19, insulin degrading enzyme, Insulin protease, Insulin proteinase, Insulin-degrading enzyme, Insulin-degrading neutral proteinase, Insulin-glucagon protease, Insulin-specific protease, Insulinase, Insulysin, Metalloinsulinase, More, pitrilysin metallopeptidase 1, Pitrm1

ECTree

     3 Hydrolases
         3.4 Acting on peptide bonds (peptidases)
             3.4.24 Metalloendopeptidases
                3.4.24.56 insulysin

Engineering

Engineering on EC 3.4.24.56 - insulysin

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A140D
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
A140F
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
A140K
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
A140N
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
A140W
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
A140Y
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
C819A
-
thiol-directed modification of C819 likely causes local structure perturbation to reduce substrate binding and catalysis
D426A
mutation diminishes activity
D426C/K899C
E107Q
catalytically inactive mutant
E110Q
-
structure comparison to wild-type enzyme structure
E111F
-
a mixed dimer in which one subunit contains the wild-type sequence and the other contains a E111F mutation that permits substrate binding, but not catalysis (E111F), exhibits a decrease in turnover number. A mixed dimer consisting of IDE:mutant E111F/Y609F IDE shows a high reduction in kcat, Km (Abz-GGFLRKHGQ-EDDnp) is 66% reduced compared to wild-type. Mixed dimer IDE:mutant E111F in which the inactive subunit can bind substrate exhibites a decreased activity than wild-type IDE towards substrate amyloid beta peptide
E111Q
E341A
-
mutant is active in degrading substrate V, relative activity closed to wild-type
E341K
-
mutant is active in degrading substrate V, relative activity 15% compared to wild-type. Exosite mutant is unable to further degrade the Ub1-74 fragment
E341Q
-
mutant is active in degrading substrate V, relative activity 20% compared to wild-type. Exosite mutant is unable to further degrade the Ub1-74 fragment
F530A
the mutation renders the enzyme hyperactive, with up to a 20fold enhancement in degrading activity
F807A
mutation decreass the Km-value of the amyloid beta substrate
G339P
-
mutant is active in degrading substrate V, relative activity 75% compared to wild-type. Exosite mutant is unable to further degrade the Ub1-74 fragment
G361A/G362A
the mutant has reduced enzymatic activity
G361P
-
mutant is active in degrading substrate V, relative activity 80% compared to wild-type. Exosite mutant is unable to further degrade the Ub1-74 fragment
H112Q
-
a mixed dimer composed of one wild-type subunit and the other subunit containing a H112Q mutation that neither permits substrate binding nor catalysis exhibits the same turnover number per active subunit as wild-type IDE. Mixed oligomer IDE:IDE mutant H112Q shows similar kcat and Km (Abz-GGFLRKHGQ-EDDnp) compared to wild-type. Mixed dimer IDE:mutant H112Q IDE in which the inactive subunit does not bind substrate exhibits a slightly higher activity than wild-type IDE towards substrate amyloid beta peptide
K364A
mutation does not change the activity
K898A
N184C/Q828C
-
30-40fold increase in activity compared to wild-type
P284G
the mutation results in a slight reduction of enzyme catalysis
P286G
the mutation results in a significant reduction of enzyme catalysis
P289G
the mutant shows wild type activity
P292G
the mutation results in intermediate reduction of enzyme catalysis
R183a
about 35% of the activity compared to wild-type enzyme with the substrate
R183D
less than 5% of the activity compared to wild-type enzyme with the substrate (7-methoxycoumarin-4-yl)acetyl-KLVFFAEDK(Dnp)-OH
R183E
about less than 5% of the activity compared to wild-type enzyme with the substrate (7-methoxycoumarin-4-yl)acetyl-KLVFFAEDK(Dnp)-OH
R183K
about 30% of the activity compared to wild-type enzyme with the substrate (7-methoxycoumarin-4-yl)acetyl-KLVFFAEDK(Dnp)-OH
R183N
about 10% of the activity compared to wild-type enzyme with the substrate (7-methoxycoumarin-4-yl)acetyl-KLVFFAEDK(Dnp)-OH
R183Q
mutant Pitrm1 R183Q is implicated in inherited amyloidogenic neuropathy. Recombinant R183Q mutant is less active than the recombinant wild-type enzyme against recombinant amyloid beta-peptide (Abeta1-40). R183Q mutant enzyme exhibits significantly decreased rate of fluorogenic peptide hydrolysis ((7-methoxycoumarin-4-yl)acetyl-KLVFFAEDK(Dnp)-OH), yet retains similar binding affinity by comparison with the wild-type enzyme. Residue R183 is positioned within an N-terminal strand-loop-strand motif that is essential for enzyme function. A requirement for charged residues within 4.5 A of residue R183 is demonstrated. The R183Q mutant enzyme exhibits increased sensitivity to heat inactivation
R767A
the mutant exists mostly as a monomer
S132C/E817C
S137A
mutation decreass the Km-value of the amyloid beta substrate
Y496A
the mutation dramatically impairs the enzymatic activity
Y609F
-
mutation Y609F in the distal part of the substrate binding site of the active subunit blocks allosteric activation regardless of the activity of the other subunit. A mixed dimer consisting of mutant Y609F IDE: mutant E111F IDE shows a high reduction in kcat and a reduction in Km compared to wild-type. A mixed dimer consisting of mutant Y609F IDE: mutant E111F/Y609F IDE shows a high reduction in kcat, Km (Abz-GGFLRKHGQ-EDDnp) is 50% reduced compared to wild-type. Substrate amyloid beta peptide: When the distal site is mutated on both subunits (Y609F IDE:IDE Y609F) there is an even greater decrease in the reaction rate
Y831A
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
Y831D
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
Y831F
Y831K
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
Y831N
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
Y831W
specific activity with the substrates (7-methoxycoumarin-4-yl)acetyl-VEALYLVCGEK(2,4-dinitrophenyl)-OH and (7-methoxycoumarin-4-yl)acetyl-QKLVFFAEDVK(2,4-dinitrophenyl)-OH is below 1 nmol/min*mg
E111A
-
change in the substrate response from sigmoidal to hyperbolic. Activating effect of ATP or triphosphate with substrate 2-aminobenzoyl-GGFLRKHGQ-(N-(2,4-dinitrophenyl)ethylenediamine) on wild-type is dampened. Mutant recognizes additional cleavage sites in substrate beta-endorphin
E111F
E111L
-
change in the substrate response from sigmoidal to hyperbolic. Activating effect of ATP or triphosphate with substrate 2-aminobenzoyl-GGFLRKHGQ-(N-(2,4-dinitrophenyl)ethylenediamine) on wild-type is dampened. Mutant recognizes additional cleavage sites in substrate beta-endorphin
E111Q
-
inactive mutant
E111V
-
change in the substrate response from sigmoidal to hyperbolic. Activating effect of ATP or triphosphate with substrate 2-aminobenzoyl-GGFLRKHGQ-(N-(2,4-dinitrophenyl)ethylenediamine) on wild-type is dampened. Mutant recognizes additional cleavage sites in substrate beta-endorphin
E768A
-
naturally occuring mutation of IDE
H112D
-
change in the substrate response from sigmoidal to hyperbolic. Activating effect of ATP or triphosphate with substrate 2-aminobenzoyl-GGFLRKHGQ-(N-(2,4-dinitrophenyl)ethylenediamine) on wild-type is changed to inhibition. Affinity to Zn2+ is lower than in wild-type
H112Q
-
change in the substrate response from sigmoidal to hyperbolic. Activating effect of ATP or triphosphate with substrate 2-aminobenzoyl-GGFLRKHGQ-(N-(2,4-dinitrophenyl)ethylenediamine) on wild-type is changed to inhibition. Affinity to Zn2+ is lower than in wild-type
H18R/A890V
I374S
-
mutation in the distal site eliminates allosterism, Vmax decreased approximately 10fold compared to wild-type, Km (Abz-GGFLRKHGQ-EDDnp) roughly similar to wild-type, no heterotropic activation with bradykinin, no significant activation through ATP
K898A/K899A/S901A
R429S
-
mutant shows greatly decreased activation by the polyphosphate anions ATP and PPP. kcat (Abz-GGFLRKHGQ-EDDnp) comparable to wild-type and Km (Abz-GGFLRKHGQ-EDDnp) higher than wild-type
V360S
-
mutation in the distal site eliminates allosterism, Vmax decreased approximately 10fold compared to wild-type, Km (Abz-GGFLRKHGQ-EDDnp) roughly similar to wild-type, no heterotropic activation with bradykinin, ATP activation is reduced to 8fold
Y248C
-
naturally occuring mutation of IDE
Y609F
additional information