The aminopeptidase from Aeromonas proteolytica (AAP) is uncompetitively inhibited by fluoride ion at pH 8.0 with an inhibition constant (Ki) of 30 mM. Thus, fluoride inactivates AAP only after substrate binding, and only a single fluoride ion binds to AAP. On the other hand, chloride ion does not inhibit AAP up to concentrations of 2 M at pH 8.0. The pH dependence of fluoride inhibition of AAP was measured over the pH range 6.0-9.5. Between pH values of 6.0 and 9.0, fluoride ion acts as a pure uncompetitive inhibitor of AAP, and the Ki increases from 1.2 to 370 mM. From a plot of pKi vs pH, a pKa value of 7.0 +/- 0.3 was extracted which corresponds to a single deprotonation process. At pH values higher than 9.0, the fluoride inhibition pattern changes to competitive. This change in inhibition pattern was attributed to a change in ionic strength or perhaps pH of the solution since fluoride ion was also found to become a competitive inhibitor of AAP at pH 8.0 in the presence of 2 M NaCl. These data, taken together with previous kinetic studies of mono- and dinuclear hydrolases with fluoride ion, suggest that a Zn(II)-bound water/hydroxide exists at the dimetal active site of AAP with a pKa of 7.0 and that this water/hydroxide acts as the active site nucleophile. The hydrolysis of L-leucine-p-nitroanilide was measured spectrophotometrically in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 5 to 800 microM. From these data, Km values were derived at each temperature studied and were found to increase exponentially with increasing temperature. Moreover, the calculated Vmax values were also found to increase over this temperature range, mimicking the Km values. An Arrhenius plot was constructed from k(cat) values and was found to be linear over the temperature range 25-85 degrees C, indicating that the rate-limiting step in AAP peptide hydrolysis is product formation and does not change as a function of temperature. From the slope of the line, the activation energy (Ea) was calculated to be 36.5 kJ/mol. The enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-358 K were found to be 34.0 kJ/mol and -94.2 J/(mol x K), respectively. The free energy of activation at 25 degrees C was found to be 62.1 kJ/mol. Combination of the available X-ray crystallographic data with the present kinetic and thermodynamic results, as well as the previously reported kinetic and spectroscopic data, has allowed a detailed catalytic mechanism for AAP to be proposed.

The nature of the interaction of the transition-state analogue inhibitor L-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with a K(i) of 6.6 microM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions. The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 A resolution. The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent. Thus, LPA binds to the dinuclear active site of AAP as an eta-1,2-mu-phosphonate with one ligand to the second metal ion provided by the N-terminal amine. A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases. On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed.

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM. The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase. Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol. The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.

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