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An intracellular aminopeptidase from Streptomyces rimosus that prefers basic amino acids
L Vitale1, I Skrtić, M Abramić
1Department of Organic Chemistry and Biochemistry, Ruder Bosković Institute, Bijenicka c. 54, 10001 Zagreb, Croatia.
Abstract:
An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt. approx. 83,000), with optimal activity at pH 7.1-7.8 and at 35-41 degrees C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM o-phenanthroline; the activity was restored upon addition of 0.05 mM Co2+, Zn2+, or Ni2+. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 microM-1 s-1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P1 and/or P1' position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified as an Arg(Lys)-preferring metalloaminopeptidase.
Insights
This study isolated a novel aminopeptidase from Streptomyces rimosus, identifying it as a metalloenzyme preferring basic amino acids. This finding distinguishes it from other known aminopeptidases.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Streptomyces rimosus is a source of various bioactive compounds, including enzymes.
- Aminopeptidases play crucial roles in cellular protein metabolism and have diverse industrial applications.
- Characterizing novel enzymes aids in understanding biological pathways and developing new biotechnological tools.
Purpose of the Study:
- To isolate and characterize an intracellular aminopeptidase from Streptomyces rimosus.
- To determine the enzyme's biochemical properties, substrate specificity, and cofactor requirements.
- To compare the novel enzyme with other known aminopeptidases from the same organism.
Main Methods:
- Electrophoretic isolation of homogeneous aminopeptidase.
- Determination of enzyme kinetics, optimal pH, temperature, and molecular weight.
- Inhibition studies using chelating agents and specific inhibitors.
- Substrate specificity analysis using amino-acid-2-naphthylamides and various peptides.
Main Results:
- An electrophoretically homogeneous, monomeric, acidic aminopeptidase (pI 4.4, MW ~83,000) was isolated.
- Optimal activity was observed at pH 7.1-7.8 and 35-41°C.
- The enzyme is a metalloaminopeptidase, inhibited by EDTA and o-phenanthroline, with activity restored by Co2+, Zn2+, or Ni2+.
- Substrate specificity revealed a preference for Arg- and Lys-2-naphthylamide and peptides with basic or bulky hydrophobic residues at P1/P1' positions.
Conclusions:
- The isolated aminopeptidase is distinct from the extracellular leucine aminopeptidase of S. rimosus.
- The enzyme can be classified as an Arg(Lys)-preferring metalloaminopeptidase.
- Its unique properties suggest potential roles in intracellular protein processing and specific biotechnological applications.