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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 °C at eight substrate concentrations ranging from 5 to 800 μM. 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 kcat values and was found to be linear over the temperature range 25−85 °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 °C calculated over the temperature range 298−358 K were found to be 34.0 kJ/mol and −94.2 J/(mol·K), respectively. The free energy of activation at 25 °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.


Accepted version. Biochemistry, Vol. 36, No. 14 (April 8, 1997): 4278-4286. DOI. © 1997 American Chemical Society Publications. Used with permission.

Richard Holz was affiliated with the Utah State University at the time of publication.

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