Isolation and characterization of an aminopeptidase from Lactobacillus acidophilus R-26.
An intracellular aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) isolated from cell extracts of Lactobacillus acidophilus R-26 was purified 634-fold to homogeneity. This enzyme, which was responsible for all of the N-terminal exopeptidase and amidase activities observed in crude extracts, had no detectable endopeptidase or esterase activity. Although a broad range of L-amino acid peptide, amide and p-nitroanilide derivatives possessing free alpha-amino termini are attacked, the enzyme favored substrates with hydrophobic N-terminal R groups. The native enzyme, which was found to be a tetramer of molecular weight 156000, contained 4 mol of tightly bound Zn2+. The catalytically inactive native zinc metalloenzyme was capable of being activated by either Zn2+, Co2+, Ni2+ or Mn2+. The shape of the log Vmax versus pH plot indicates that two active-center ionizable groups (pKES1 = 5.80; pKES2 = 8.00) may be involved in catalysis. 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 corresponds to the modification of one histidine residue per monomer of protein.
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000302:isolation & purification*; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007779:Lactobacillus acidophilus / Q000201:enzymology*; D008751:Methylene Blue; D008970:Molecular Weight; D010777:Photochemistry; D012117:Resins, Synthetic; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000596:Amino Acids; D002413:Cations, Divalent; D002614:Chelating Agents; D012117:Resins, Synthetic; D013439:Sulfhydryl Reagents; C024997:Chelex 100; D000626:Aminopeptidases; D008751:Methylene Blue