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Literature summary for 3.4.22.34 extracted from

  • Dall, E.; Brandstetter, H.
    Activation of legumain involves proteolytic and conformational events, resulting in a context- and substrate-dependent activity profile (2012), Acta Crystallogr. Sect. F, 68, 24-31.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
His-tagged fusion protein stably transfected in LEXSY P10 host strain Homo sapiens

Crystallization (Commentary)

Crystallization (Comment) Organism
super-activated legumain is crystallized in complex with the legumain-specific inhibitor Ala-Ala-Asn-chloromethylketone to resolution of 2.47 A Homo sapiens

Protein Variants

Protein Variants Comment Organism
C189S dead mutant: in contrast to the wild-type protein, no evidence for the additional C-terminal cleavage after Asp303/309 is found Homo sapiens
D303E/D309E double mutant in which the pH 4-dependent cleavage site is disrupted Homo sapiens
DELTA18-25 N-terminal truncation variant lacking the N-terminal propeptide [DELTA-(Val18-Asp25)] is created. The activity of this variant resembles that of the wild-type protein and it is inactive in the unprocessed form (Gly26-Tyr433) at pH 7.0 or pH 5.5. Like the wild-type protein, the truncation variant undergoes correct maturation at pH 5.5 to the Gly26-Asn323 form, confirming correct folding of the protein Homo sapiens

Inhibitors

Inhibitors Comment Organism Structure
Ac-Tyr-Val-Ala-Asp-chloromethylketone the rate of inhibition by Asp-CMK is approximately 3times more rapid at pH 4.0 than at pH 5.5 Homo sapiens
Ala-Ala-Asn-chloromethylketone the rate of inhibition by Asn-CMK is approximately 1.3times more rapid at pH 5.5 than at pH 4.0 Homo sapiens

Organism

Organism UniProt Comment Textmining
Homo sapiens Q99538
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
proteolytic modification legumain is synthesized as a zymogen und undergoes pH-dependent autoproteolytic activation. All but one of the autocatalytic cleavage events occur in trans, with only the release of the C-terminal propeptide being relevant to enzymatic activity. Legumain matures to the fully active form (super-activation) only when incubated at pH 4.0 Homo sapiens

Purification (Commentary)

Purification (Comment) Organism
using Ni–NTA chromatography Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
Ac-Tyr-Val-Ala-Asp-4-nitroanilide + H2O
-
Homo sapiens ?
-
?
benzoyl-L-Arg-4-nitroanilide + H2O
-
Homo sapiens ?
-
?

Synonyms

Synonyms Comment Organism
legumain
-
Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
additional information
-
legumain activity is pH-dependent and substrate-dependent Homo sapiens
4
-
Legumain activity shows a pH optimum of 4.0 towards substrates with Asp at the P1 position Homo sapiens
4
-
superactivation of prolegumain requires its incubation at pH 4.0 Homo sapiens
5.5
-
Legumain activity shows a pH optimum of 5.5 towards substrates with Asn at the P1 position: maximum activity towards substrate benzoyl-L-Arg-4-nitroanilide is observed at pH 5.5 Homo sapiens

pH Range

pH Minimum pH Maximum Comment Organism
5.5 7 at neutral pH, full-length legumain remains stable with no indication of proteolytic processing. Shifting the pH to 5.5 the C-terminal propeptide Asp324-Tyr433 is cleaved within 20 h at 37°C Homo sapiens

pH Stability

pH Stability pH Stability Maximum Comment Organism
4 5.5 the thermal stability of active legumain is highest in the pH interval 4.0-5.5, with a melting temperature Tm of 60°C. At pH values higher than 6.0 the melting temperature Tm decreased to below 30°C Homo sapiens