Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans

• Published in: Biochimica et biophysica acta Vol. 800; no. 2; pp. 127 - 134
• Main Author: Machuga, Edward J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 30.07.1984
• Subjects: Amino Acids - analysis; Aminopeptidase; Aminopeptidases - isolation & purification; Dialysis; Dipeptides - metabolism; Edetic Acid - pharmacology; Ethylmaleimide - pharmacology; Exopeptidase; Hydrogen Peroxide - pharmacology; Hydrogen-Ion Concentration; Mercaptoethanol - pharmacology; Molecular Weight; Photochemistry; S. durans; Streptococcus - enzymology; Substrate Specificity; Exopeptidase; Aminopeptidase; S. durans
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(84)90050-3

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 μmol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM β-mercaptoethanol. The purified aminopeptidase ( M r 300 000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49 400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of L - alanyl-p- nitroanilide exhibited a bell-shaped pH dependence for log V max/ K m(p K 1 = 6.35; p K 2 = 8.50) while the log V max versus pH profile showed only an acid limb (p K = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity.