Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans.
An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 mumol/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 beta-mercaptoethanol. The purified aminopeptidase (Mr 300000) 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 49400, 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 Vmax/Km (pK1 = 6.35; pK2 = 8.50) while the log Vmax versus pH profile showed only an acid limb (pK = 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.
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000302:isolation & purification*; D003956:Dialysis; D004151:Dipeptides / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D010777:Photochemistry; D013291:Streptococcus / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D004151:Dipeptides; D008623:Mercaptoethanol; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D000626:Aminopeptidases; D005033:Ethylmaleimide