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Literature summary for 3.4.11.5 extracted from

  • Zdunek-Zastocka, E.; Grabowska, A.; Branicki, T.; Michniewska, B.
    Biochemical characterization of the triticale TsPAP1, a new type of plant prolyl aminopeptidase, and its impact on proline content and flowering time in transgenic Arabidopsis plants (2017), Plant Physiol. Biochem., 116, 18-26 .
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
2-mercaptoethanol 3.9fold activation at 1.0 mM Secale cereale x Triticum turgidum subsp. durum
DTT 5.7fold activation at 0.1 mM Secale cereale x Triticum turgidum subsp. durum
EDTA 6.13fold activation at 1.0 mM Secale cereale x Triticum turgidum subsp. durum

Cloned(Commentary)

Cloned (Comment) Organism
gene pap1, recombinant expression of His-tagged enzyme in Escherichia coli, recombinant expression in transgenic Arabidopsis thaliana plants in rosette leaves Secale cereale x Triticum turgidum subsp. durum

Protein Variants

Protein Variants Comment Organism
additional information impact of TsPAP1 overexpression on flowering time and the number of siliques due to the enhanced accumulation of proline in transgenic Arabidopsis thaliana plants. The recombinant TsPAP1 protein is expressed without the signal peptide, which could have a negative impact on its activity Secale cereale x Triticum turgidum subsp. durum

Inhibitors

Inhibitors Comment Organism Structure
Cd2+ 94% inhibition at 0.0005 mM Secale cereale x Triticum turgidum subsp. durum
Co2+ 95% inhibition at 0.05 mM Secale cereale x Triticum turgidum subsp. durum
Cu2+ 96% inhibition at 0.005 mM Secale cereale x Triticum turgidum subsp. durum
diisopropyl fluorophosphate 87% inhibition at 0.1 mM, 94% at 1.0 mM Secale cereale x Triticum turgidum subsp. durum
Fe2+ 29% inhibition at 0.5 mM Secale cereale x Triticum turgidum subsp. durum
iodoacetamide slight inhibition at 1 mM, 90% inhibition at 10 mM Secale cereale x Triticum turgidum subsp. durum
additional information no inhibition by E64, i.e. trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, and pepstatin A at 10 mM Secale cereale x Triticum turgidum subsp. durum
PMSF 72% inhibition at 0.1 mM, 92% at 1.0 mM Secale cereale x Triticum turgidum subsp. durum
Zn2+ 97% inhibition at 0.0005 mM Secale cereale x Triticum turgidum subsp. durum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0775
-
Pro-beta-naphthylamide pH 7.5, 25°C, recombinant enzyme Secale cereale x Triticum turgidum subsp. durum
0.28
-
Ala-beta-naphthylamide pH 7.5, 25°C, recombinant enzyme Secale cereale x Triticum turgidum subsp. durum

Localization

Localization Comment Organism GeneOntology No. Textmining
chloroplast
-
Secale cereale x Triticum turgidum subsp. durum 9507
-
cytoplasm
-
Secale cereale x Triticum turgidum subsp. durum 5737
-

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ activates slightly at 0.5 mM Secale cereale x Triticum turgidum subsp. durum
additional information no effect by Mg2+, Na+, Ba2+, Mn2+ at 0.5 mM Secale cereale x Triticum turgidum subsp. durum

Organism

Organism UniProt Comment Textmining
Secale cereale x Triticum turgidum subsp. durum G9J616 triticosecale
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography Secale cereale x Triticum turgidum subsp. durum

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
Ala-beta-naphthylamide + H2O
-
Secale cereale x Triticum turgidum subsp. durum Ala + beta-naphthylamine
-
?
Hyp-beta-naphthylamide + H2O
-
Secale cereale x Triticum turgidum subsp. durum Hyp + beta-naphthylamine
-
?
additional information the recombinant enzyme shows preference for substrates with a proline at the N-terminus. The enzyme also hydrolyzes beta-naphthylamides of hydroxyproline and alanine, although the observed activity is almost 2fold lower than against Pro-beta-naphthylamide. The activity against the other amino acid-beta-naphthylamides tested (with Phe-, Glu-, Arg-, Tyr-, Leu-, Asp-, Met-, Trp-, and Val-beta-naphthylamide) is not detectable or does not exceed 7% of the maximal activity. Among the peptides with proline at the N-terminus, TsPAP1 shows a much higher preference for dipeptides than tri- and tetrapeptides. Not only the length of the peptide is important, as also the amino acid in the Y position influences the rate of proline liberation. Among dipeptides, the most preferred is Pro-Gly and Pro-Pro while Pro-Ala is hydrolyzed at only 10% the rate of Pro-Gly. The activity against tripeptide Pro-Gly-Gly is 27% of the maximal activity while the rate of hydrolysis of the tetrapeptides is very low and does not exceed 10% of that against Pro-Gly Secale cereale x Triticum turgidum subsp. durum ?
-
?
Pro-Ala + H2O low activity Secale cereale x Triticum turgidum subsp. durum Pro + Ala
-
?
Pro-beta-naphthylamide + H2O preferred substrate Secale cereale x Triticum turgidum subsp. durum Pro + beta-naphthylamine
-
?
Pro-Gly + H2O
-
Secale cereale x Triticum turgidum subsp. durum Pro + Gly
-
?
Pro-Gly-Gly + H2O low activity Secale cereale x Triticum turgidum subsp. durum Pro + Gly-Gly
-
?
Pro-Gly-Gly-Gly + H2O low activity Secale cereale x Triticum turgidum subsp. durum Pro + Gly-Gly-Gly
-
?
Pro-Leu + H2O low activity Secale cereale x Triticum turgidum subsp. durum Pro + Leu
-
?
Pro-Leu-Gly + H2O low activity Secale cereale x Triticum turgidum subsp. durum Pro + Leu-Gly
-
?
Pro-Pro + H2O
-
Secale cereale x Triticum turgidum subsp. durum Pro + Pro
-
?

Subunits

Subunits Comment Organism
monomer
-
Secale cereale x Triticum turgidum subsp. durum

Synonyms

Synonyms Comment Organism
Prolyl aminopeptidase
-
Secale cereale x Triticum turgidum subsp. durum
TsPAP1
-
Secale cereale x Triticum turgidum subsp. durum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
recombinant enzyme, without EDTA Secale cereale x Triticum turgidum subsp. durum
37
-
recombinant enzyme, with EDTA Secale cereale x Triticum turgidum subsp. durum

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
-
60 activity range, profile overview Secale cereale x Triticum turgidum subsp. durum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
recombinant enzyme Secale cereale x Triticum turgidum subsp. durum

pH Range

pH Minimum pH Maximum Comment Organism
6 9.2 activity range, profile overview Secale cereale x Triticum turgidum subsp. durum

General Information

General Information Comment Organism
evolution enzyme TsPAP1 belongs to the S33.001 subfamily of aminopeptidases with presence of additional tens of amino acids at the N-terminus of the most plant sequences but not of microorganisms. These additional amino acids constitute a potential signal peptide sequence most likely directing the proteins to chloroplasts. The plant PAP sequences display a high percent of amino acid identity from only the second methionine from the N-terminus (M86 in TsPAP1) while not within the sequence of the signal peptide Secale cereale x Triticum turgidum subsp. durum
malfunction impact of TsPAP1 overexpression on flowering time and the number of siliques due to the enhanced accumulation of proline in transgenic Arabidopsis thaliana plants, phenotype, overview Secale cereale x Triticum turgidum subsp. durum