Xaa-Pro-dipeptidyl-aminopeptidase from Lactococcus lactis catalyses kinetically controlled synthesis of peptide bonds involving proline.

Xaa-Pro-dipeptidyl-aminopeptidase (EC 3.4.14.5) from Lactococcus lactis (PepX) was used, for the first time, as a catalyst in kinetically controlled synthesis of peptide bonds involving proline. PepX had amidase and esterase activities in addition to peptidase activity. Thus amide and ester derivatives of X-Pro peptides could be employed as acyl donors. PepX showed a broad specificity for the residue in position P'1, accepting a large variety of amino acid amides, esters, peptides as well as free amino acids as nucleophiles. This also indicated that it was not necessary to protect the C-terminus of the nucleophile. The major factors controlling yield, e.g. pH, an excess of nucleophile, ionic strength and type of carboxyl protecting and activating groups, were evaluated. Under optimum reaction conditions (pH 8.5, high excess of nucleophile over acyl donor and moderate ionic strength) the selectivity of the reaction ranged from 5 to 99% depending on the structure of the nucleophile and the acyl donor. Our work contributes to the elucidatation of the mechanism of aminolysis reactions catalysed by an aminopeptidase.

Reversible enzymic protection of the alpha-amino group of amino acid derivatives using an aminopeptidase A.

In previous papers we have reported that an aminopeptidase A (EC 3.4.11.7) purified from Staphylococcus chromogenes was able to catalyse the introduction of L-malic acid at the N-terminus of Tyr and Phe derivatives. We now show that this enzyme can be used for selective alpha-amino protection of derivatives of probably all amino acids, except Gly and Pro, by the malyl group. The following L-malyl derivatives were synthesized in thermodynamically controlled reactions with yields ranging from 4 to 47%: L-malyl-Tyr-OEt, -ALA-OMe, -Ser-OEt, -Lys-OEt, -Phe-OMe, -Met-NH2, -Glu-MH2, Arg-NH2, -Tye-NH2, -Val-NH2, -Ala-Phe and -Ala-Phe-NH2 (OEt and OMe are ethyl and methyl esters respectively). The reactions were monitored by reverse-phase h.p.l.c.; the products were quantified by amino acid analysis, and their structure was confirmed by m.s. No synthesis was obtained with Gly and Pro derivatives as nucleophiles. The effects of pH, temperature, enzyme concentration, nucleophile concentration, reaction time and addition of an organic co-solvent were studied. An important shift towards synthesis was obtained by carrying out the reactions at 55 degrees C in the presence of 55% organic co-solvent Triglyme (2,4,8,11-tetraoxadodecane) [8-60-fold increase in Ksyn. ([product] [acyl-donor]-1 [nucleophile]-1)].

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.