Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
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 ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.