Aminopeptidase yscCo‐II: a new cobalt‐dependent aminopeptidase from yeast—purification and biochemical characterization

Herrera‐Camacho, Irma; Morales‐Monterrosas, Rosalva; Quiróz‐Alvarez, Rubén

doi:10.1002/(sici)1097-0061(200002)16:3<219::aid-yea523>3.3.co;2-a

Cited by 3 publications

(12 citation statements)

References 21 publications

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“…This property is common to several aminopeptidases from ascomycetes [12,13,16,17,26,27] and basidiomycetes [19,20]. As has been reported for other fungal aminopeptidases [16,17], the presence of blocking agents from serin proteases, such as Pefabloc, PMSF and leupeptin, inhibited partially the activity suggesting that serine residues might be participating in the catalysis of this enzyme. As has been reported for other fungal aminopeptidases [16,17], the presence of blocking agents from serin proteases, such as Pefabloc, PMSF and leupeptin, inhibited partially the activity suggesting that serine residues might be participating in the catalysis of this enzyme.…”

Section: Substratesupporting

confidence: 67%

“…This property is common to several aminopeptidases from ascomycetes [12,13,16,17,26,27] and basidiomycetes [19,20]. This property is common to several aminopeptidases from ascomycetes [12,13,16,17,26,27] and basidiomycetes [19,20].…”

Section: Substratementioning

confidence: 90%

“…The described ascomycete and basidiomycete aminopeptidases include monomeric [20,26], dimeric [12] and multimeric [16,[18][19][20] forms with subunits varying from 30 to 92 kDa. The described ascomycete and basidiomycete aminopeptidases include monomeric [20,26], dimeric [12] and multimeric [16,[18][19][20] forms with subunits varying from 30 to 92 kDa.…”

Section: Substratementioning

confidence: 99%

“…In contrast, a methionine aminopeptidase from S. cerevisiae has an alkaline isoelectric point (pI 7.8) [26]. Similarly to other intracellular fungal aminopeptidases [13,16,19], pumAPE exhibits a neutral optimal pH of enzyme activity with a narrow range of activity. An exception is the aminopeptidase from G. frondosa with an alkaline optimal pH of enzyme activity (pH 8.5) [20].…”

Section: Substratementioning

confidence: 99%

“…Methionine aminopeptidases from S. cerevisiae, encoded by the MAP1 and MAP2 genes, remove the N-terminal methionine of newly synthesized proteins [9]. However, most ascomycete aminopeptidases have no associated function evidenced through genetic or biochemical studies [12][13][14][15][16][17]. However, most ascomycete aminopeptidases have no associated function evidenced through genetic or biochemical studies [12][13][14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

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Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

Mercado-Flores

Noriega-Reyes

Ramı́rez-Zavala

et al. 2004

FEMS Microbiology Letters

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The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 degrees C toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.

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Section: Substratesupporting

confidence: 67%

Section: Substratementioning

confidence: 90%

Section: Substratementioning

confidence: 99%

Section: Substratementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

Mercado-Flores

Noriega-Reyes

Ramı́rez-Zavala

et al. 2004

FEMS Microbiology Letters

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Purification and characterization of a lysine aminopeptidase fromKluyveromyces marxianus

RamÃrez-Zavala

Mercado-Flores

HernÃ¡ndez-RodrÃguez³

et al. 2004

FEMS Microbiology Letters

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A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4-8, its optimal temperature was 45 degrees C and the enzyme became unstable at temperatures above 55 degrees C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and Vmax for L-lysine-p-nitroanilide were 0.33 mM and 2.2 mM min(-1) per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.

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Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from Schizosaccharomyces pombe

et al. 2007

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Leucyl aminopeptidases (LAPs; EC 3.4.11.1) are members of the M17 family of metallo-peptidases [1] that cleave leucine residues from the N-terminus of proteins, peptides and synthetic substrates, although substantial activity may also be evident for other amino acids. They are widely distributed in organisms from bacteria to humans, the best characterized being those from Bos taurus, Escherichia coli and Lycopersicon esculentum (tomato) [2][3][4]. X-ray crystal structures have shown that these enzymes exist as homohexamers, comprising two trimers stacked on top of one another. In many cases, each monomer binds two zinc ions and folds into two a ⁄ b-type quasi-spherical globular domains, producing a comma-like shape [5] A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS ⁄ PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl-4-nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N-terminal amino acids. A clear dependence on Mn 2+ concentration for activity was found, and an apparent association constant of 0.33 mm was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (K i ¼ 0.14 lm), while chelating agents such as chloroquine, EDTA and 1,10-phenanthroline also reduced enzyme activity. A MALDI-MS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C-terminal catalytic domain showing a characteristic topology of eight b-sheets surrounded by a-helical segments in the form of a saddle.Abbreviation LAP, leucyl aminopeptidase; Lys-NA, lysine 4-nitroanilide.

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Aminopeptidase yscCo‐II: a new cobalt‐dependent aminopeptidase from yeast—purification and biochemical characterization

Cited by 3 publications

References 21 publications

Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

Purification and characterization of a lysine aminopeptidase fromKluyveromyces marxianus

Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from Schizosaccharomyces pombe

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