Specificity of Aeromonas Aminopeptidase toward Oligopeptides and Polypeptides

Abstract

A variety of oligopeptides and polypeptides was subjected to the action of Aeromonas aminopeptidase and the sequential release of amino acid residues was determined quantitatively. The residues that proved susceptible to hydrolysis included all hydrophobic, aromatic and basic amino acids plus proline. Aspartyl, glutamyl and cysteic acid residues were not removed from the aminoterminus of any substrate tested, even at high enzyme concentrations but in contrast, asparagine, glutamine and aminoethylated cysteine were released. Glycine was generally resistant to hydrolysis, but was slowly released from some substrates dependent upon the adjacent residues. The specificity and ease of purification of this aminopeptidase should make it a useful tool for sequence determination.Substrate specificities of newly discovered aminopeptidases have been determined largely by assessment of their action toward amino acid amides and esters, and toward di-and tripeptides [l-51. The current availability of oligopeptides and polypeptides of known sequences, however, makes it possible to study the specificity of various proteolytic enzymes, including aminopeptidases, toward substrates which more closely resemble those found in nature. The use of such substrates should permit one to evaluate a new enzyme for its potential usefulness in sequence determinations. In previous communications we have described the isolation and general properties of an extracellular aminopeptidase from the marine bacterium Aeromonas proteolytica [6--8]. Unlike a number of aminopeptidases from other sources, the Aermonas enzyme is monomeric and, with a molecular weight of 29500, it is the smallest aminopeptidase thus far described in the literature. Although this enzyme is a zinc metalloprotein, it requires no addition of ions for full activity and it possesses a high degree of thermal stability. Our previous results, obtained with amino acid amide and dipeptide substrates [%], indicated that Aeromonas aminopeptidase has a restricted substrate specificity and it appeared desirable to test its activity toward a number of oligopeptides and polypeptides of known sequence, to delineate more fully its substrate specificity. The purpose of the studies reported herein was to determine quantitatively the rate of release of various amino acid residues from such substrates with a view to assessing the potential usefulness of Enzyme. Aminopeptidase (EC 3.4.1.2).Aeromonas aminopeptidase as a reagent for sequencing oligopeptides derived from the partial cleavage of proteins. EXPERIMENTAL PROCEDURE MaterialsAeromonas aminopeptidase was isolated by the procedure described previously [7] and each preparation was shown to be homogeneous by polyacrylamidc-gel electrophoresis at pH 8.6 according to the method of Davis [9]. The specific activity of these preparations was 350-400 units per milligram as determined by the spectrophotometric method described by Wagner et al. [%I, using 20 mM leucinamide in 50 mM Tris buffer pH 8.0. Enzyme concentrations were determined a t 278.5 nm ...

Structure of peptidase T from Salmonella typhimurium

“…The different domain organization in LAP, CPA, APP, CG2 and peptidase T may reflect different ways to discriminate against longer polypeptides. In APP and CPA, the N‐terminal (APP) or the C‐terminal (CPA) end of the substrate binds into a pocket and the absence of additional steric hindrance enables the enzyme to cleave polypeptides of varying size [31,32]. In peptidase T and CG2, however, the presence of the dimerization domain may restrict the size of the substrate on the C‐terminal side of the scissile bond.…”

“…This negative charge may also prevent dipeptides from entering the active site, as there would be electrostatic repulsion between the C‐terminal carboxylate group and this part of the enzyme. APP, which also has a negatively charged active site, does not cleave peptides with a negatively charged P1 side chain, and displays lower activity towards dipeptides and peptides with a negatively charged group in P1′ position [31,32]. This suggests that there is a penalty for having negative charge on the substrate too close to the N‐terminus.…”

Fine tuning the N-terminus of a calcium binding protein: ?-lactalbumin

“…The choice of A. proteolytica aminopeptidase was based on the fact that the activity of this enzyme is completely blocked by the amino acids with large negatively charged side chains9, 10 because the first residue in the bovine α‐LA sequence is glutamic acid. The digestion reaction was performed at a substrate:enzyme ratio of about 100:1.…”

Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation

“…To this end, GS-SplB was incubated with a roughly equimolar amount of aminopeptidase from Aeromonas proteolytica (AAP) (24), and the proteolytic activity characteristic of SplB (but not AAP) was monitored. AAP was selected for this experiment because it was reported to remove most amino-terminal residues except glutamic acid, the exact residue found at the N terminus of native, mature SplB.…”