Purification and characterization of an aminopeptidase A from Staphylococcus chromogenes and its use for the synthesis of amino-acid derivatives and dipeptides.
An aminopeptidase with original specificity was purified 3800-fold to homogeneity from a cellular extract of Staphylococcus chromogenes. The enzyme was specific for acidic amino acids (Asp and Glu) at the N-terminus of peptides and thus can be classified as an aminopeptidase A. However, its specificity was not restricted to acidic amino acids: alpha-hydroxy acids such as L-malic and L-lactic acids were also accepted in position P1. The enzyme had a broad specificity for the residue at position P' 1, accepting all types of amino acids, including Pro, in this position. The optimal conditions for the hydrolysis of Asp-Phe-NH2 were pH 9.5 and 60 degrees C. The enzyme was inhibited by chelating agents and serine-protease inhibitors. The activity lost by treatment with chelating agents could be restored by Mn2+ or Zn2+ which also stimulated the native enzyme. This suggests that it is a metalloprotease with a serine residue essential for the activity. The native enzyme had an apparent molecular mass of 430 kDa on gradient-gel electrophoresis and subunits of 43 kDa as determined by SDS/PAGE. The enzyme catalyzed the synthesis of peptide and amino acid derivatives such as Asp-Phe-OMe (Aspartame) and malyl-Tyr-OEt from L-Asp and L-malic acid as acyl donors and L-Phe-OMe and L-Tyr-OEt as nucleophiles, respectively. The use of the enzyme as a reagent in protease-catalyzed peptide synthesis, N-terminal protection and subsequent deprotection, is described.
Yoshpe-Besançon
I
I
Enzymology Unit, Bât. 224, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
Auriol
D
D
Paul
F
F
Monsan
P
P
Gripon
J C
JC
Ribadeau-Dumas
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D043384:Glutamyl Aminopeptidase; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D013210:Staphylococcus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D002413:Cations, Divalent; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase