Specificity of Aeromonas Aminopeptidase toward Amino Acid Amides and Dipeptides”

Abstract The substrate requirements of Aeromonas aminopeptidase toward NH2-terminal residues were determined by use of amides representing all classes of amino acids, and the effects of the adjacent residue on hydrolysis were assessed by use of leucyl dipeptides in which the identity of the COOH-terminal residue was varied. The enzyme was specific for substrates possessing bulky, hydrophobic residues in the NH2 terminus; of 20 amides tested, measurable rates of hydrolysis were found only for leucinamide, norleucinamide, norvalinamide, isoleucinamide, valinamide, methioninamide, and phenylalaninamide. Of these, leucinamide showed the highest ratio of κcat:Km(app). Among the dipeptides, Leu-Met had the highest κcat:Km(app) ratio and the leucyl peptides with arginine, phenylalanine, tryptophan, and tyrosine in the COOH terminus exhibited successively lower ratios. The selectivity of the aminopeptidase for large hydrophobic residues in the penultimate position was emphasized by the fact that low κcat:Km(app) ratios were exhibited toward Leu-Ala, Leu-Gly, and Leu-OMe.

The substrate requirements of Aeromonas aminopeptidase toward NH%-terminal residues were determined by use of amides representing all classes of amino acids, and the effects of the adjacent residue on hydrolysis were assessed by use of leucyl dipeptides in which the identity of the COOHterminal residue was varied.
Of these, leucinamide showed the highest ratio of kcat. *Kn(am -Among the dipeptides, Leu-Met had the highest koat. * Kmcapp) ratio and the leucyl peptides with arginine, phenylalanine, tryptophan, and tyrosine in the COOH terminus exhibited successively lower ratios. The selectivity of the aminopeptidase for large hydrophobic residues in the penultimate position was emphasized by the fact that low kcat: Km(appl ratios were exhibited toward Leu-Ala, Leu-Gly, and Leu-OMe. Evidence from previous work (1, 2) suggested that the substrate specificity of Aeromonas aminopeptidase was different from that of aminopeptidases previously described (3-8). We thus were prompted further to investigate the catalytic specificity of this enzyme which is particularly interesting because it has the lowest molecular weight (29,500) of any aminopeptidase yet characterized, does not consist of subunits, and is a zinc metalloenzyme (2). The present investigation was therefore undertaken to determine the influence exerted by the NH2terminal and penultimate residues of substrates on hydrolysis. The former was investigated by use of amino acid amides in order to assess the influence of the NHz-terminal residue, uncomplicated by other substituents, whereas the effects of penultimate residues were investigated with leucyl dipeptides in which Enzyme Assays-L-Leucinamide' was the substrate used for standard enzyme assays, and 1 unit of aminopeptidase was defined as that quantity of enzyme which catalyzed the hydrolysis of 1 pmole of leucinamide per min at pH 8.0 and 25". The hydrolysis of amide, dipeptide, and ester substrates was measured from the decrease in absorbance at 230 nm in a Gilford model 222 recording spectrophotometer, the cuvette compartment of which was maintained at 25 f 0.1"; enzyme and substrate solutions were equilibrated at this temperature prior to being used in the assays. Assays in l-cm cuvettes were performed by adding 50 ~1 of enzyme solution to 2.95 ml of substrate, both in 50 mM Tris-HCl buffer, pH 8.0; the solutions were immediately mixed and the decrease in absorbance was recorded for about 5 min. The contribution of the bond in each substrate to the molar extinction at 230 nm was determined by subtracting the sum of molar extinctions of the hydrolytic products from that of the substrate and the resulting values were used to calculate the extent of hydrolysis.
Reaction velocities were calculated from the initial slopes of tracings of absorbance against time (apparent zero order rate) and expressed as micromoles of substrate hydrolyzed per min per ml of enzyme solution.
Substrates that showed no discernible change in absorbance after 5 min of incubation with the enzyme were qualitatively evaluated by incubation with the aminopeptidase for 6 hours at 25", then subjected to high voltage electrophoresis to detect any hydrolysis. and Knscapp) (apparent K,) represent at least 30 individual assays. Calculations of Km(sgp) and Ti,,, (maximum velocity of an enzyme-substrate reaction) were made from Lineweaver-Uurk double reciprocal plots analyzed by the method of least squares. Values for kcat were obtained by dividing V,n,, by the concentration of enzyme in micromoles per ml of reaction mixture.
Other Procedures-Concentrations of aminopeptidase solutions were determined spectrophotometrically at 278 nm by use of the value EE,, = 14.4 (2). Thin layer chromatography was performed on glass plates coated with hdsorbosil 1 and activated by heating at 100" for 1 hour; the chromatograms were developed with 77 y0 ethanol.
One-dimensional high voltage electrophoresis was done as described previously (9) with a formic acid-acetic acid-mater (1: 4:45) buffer at pH 1.9 and Varsol as the coolant.

InJluence of XH&erminal
Residue-In preliminary experiments, amides representative of all classes of amino acids were tested at 20 mM concentration, and only seven, all of which contained neutral, bulky residues in the NH2 terminus, were cleaved at measurable rates. The identities of the susceptible substrates, along with all others tested, are shown in Table I. A minimum chain length is obviously required for susceptibility to the aminopeptidase as the amides of oc-aminobutyric acid, alanine, and glycine showed no activity as substrates.
Isoglutamine and isoasparagine were not hydrolyzed, probably because the negative charge on these amides prevented cleavage, but in contrast, activity could be discerned qualitatively when the positively charged amides of arginine and lysine were tested. Determination of the kinetic constants kcat and Kmcapp) for the susceptible substrates (Table I) revealed that leucinamide not only had the highest kcat value, but its kcat:Km(app) ratio was 4-fold greater than any other amide.
Bender and KBzdy (10) have proposed that the value lccat: Kmcapp) is probably always a meaningful kinetic constant in correlations of structure and specificity, and from certain simple assumptions one can show that for substrates with the same kcat, differences in their Km(8pp) values are indicative of different enzyme-substrate affinities. In both examples, the B-carbon compound was bound more effectively than the 5-carbon substrate.
The marked influence exerted by the position of a methyl group is evident from a comparison of the kinetic values for leucinamide, norleucinamide and isoleucinamide, and a comparison of the values for methioninamide and norleucinamide reveals the effect of substituting a sulfur atom for a carbon. In addition to the degree and position of branching, the size of the NH*-terminal residue affects enzyme-substrate affinity; amides less bulky than valinamide or norvalinamide are not appreciably bound, as evidenced by the fact that glycinamide, threoninamide, and cu-NHz-butyramide not only did not serve as substrates themselves, but failed to act as inhibitors of the hydrolysis of a Dipeptides not hydrolyzed by the aminopeptidase were Leu-Glu, Leu-Pro, n-Leu-L-Leu, and L-Leu-n-Leu. b Assayed in l-mm cuvettes to compensate for high absorbance. The volumes of enzyme and substrate were one-tenth those used in the standard assays. c Assayed by measuring the decrease in absorbance at 248 nm.

Leu-OMe
Phe-OMe specific, as indicated by the failure of n-leucinamide to inhibit the hydrolysis of the L enantiomorph.
InJluence of Penultimate Substituents-Values for K,,,capp) and koat of leucyl dipeptides are shown in Table II which reveals that rates of hydrolysis (k,,t) were highest for those containing aromatic residues, methionine, or arginine in the penultimate leucinamide.
Binding by the aminopeptidase is also stereo-position. the adjacent position rendered the dipeptide nonsusceptible to dues contribute the nitrogen atom of the peptide bond (12). hydrolysis, as had the presence of these residues in the NH2 Hydrolysis of a peptide chain by the endopeptidnse thus iiberates terminus.
Enantiomorphic specificity also is apparent in the a fragment having an NI-Iz-terminal residue that is particularly penultimate position as evidenced by the failure of L-Leu-n-Leu susceptible to the aminopeptidase. The 1)attern is analogous to serve as a substrate or to inhibit the hydrolysis of either to that found in bovine pancreatic juice in which chymotryptic n-leucinamide or n-Leu-n-Leu. The latter observation suggests cleavage liberates products most susceptible t,o carboxypeptidase the L-Leu-n-Leu was not bound by the catalytic site on the A and hydrolysis by trypsin yields l'referred subst,rntes for aminopeptidase.
Dipeptides generally had lower Km(app) values carboxypeptidase 13. Such complement,ary specificities betlveen than the amino acid amides, probably because the adjacent endopeptidases and exopeptidases of the same cellular origin amino acid residue enhanced substrate binding.
Differences may prove to be of frequent occurrence in nature. in enzyme-substrate affinity among the dipeptides were evident, as Leu-Met and Leu-Trp had similar keat values but differed substantially with respect to Km(itpp); a similar situation existed dipeptide tested, and the influence of the position of a methyl group on the penultimate residue is evident from a comparison of the kinetic values for Leu-Leu and Leu-Ile.
Our previous work on the isolation and characterization of Aeromonas aminopeptidase was done with Leu-NX as the substrate (1, 2) and it is interesting to compare the kinetic values of this compound with those of the dipeptides and amino acid amides. Although lc,,t for Leu-NA was not unusually high, K~am) was lower than that of any amino acid amide (Table I) and in fact, was lower than more than half of the dipeptides, probably because the bulky P-naphthylamine moiety enhanced binding to the enzyme. The methyl esters of leucine and phenylalanine were hydrolyzed at rates comparable to those shonn by a number of dipeptides, but due to its extraordinarily high J<m(app), Leu-OMe had the lowest kcat:Rm(app) ratio of any substrate tested and it is apparent that the structural differences between Leu-OMe and leucinamide exert profound effects on both binding and the catalytic process. In contrast, Phe-OAIe had a higher kcat:Kn(app) ratio than phenylalaninamide because of the low keat of the latter.
In addition to the aminopeptidase, Aeromonas proteolytica produces an extracellular neutral proteinase (11-13) with a specificity toward substrates to which bulky hydrophobic resi-