The aminopeptidase from Aeromonas proteolytica can function as an esterase

Abstract.

The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester (L-Leu-OEt) with a rate of 96±5 s^–1 and a K _m of 700 µM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67±5 s^–1. The k _cat values for the hydrolysis of L-Leu-OEt and L-leucine-p-nitroanilide (L-pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature and is product formation. The activation energy (E _a) for the activated ES^‡ ester complex is 13.7 kJ mol^–1, while the enthalpy and entropy of activation at 25 °C calculated over the temperature range 298–338 K are 11.2 kJ mol^–1 and –175 J K^–1 mol^–1, respectively. The free energy of activation at 25 °C was found to be 63.4 kJ mol^–1. The enthalpy of ionization was also measured and was found to be very similar for both peptide and ester substrates, yielding values of 20 kJ mol^–1 for L-Leu-OEt and 25 kJ mol^–1 for L-pNA. For peptide and L-amino acid ester cleavage reactions catalyzed by AAP,${{k_{cat}^{H_2 O} } \mathord{\left/ {\vphantom {{k_{cat}^{H_2 O} } {k_{cat}^{D_2 O} }}} \right. \kern-\nulldelimiterspace} {k_{cat}^{D_2 O} }} = 2.75$ and 6.07, respectively. Proton inventory data suggest that two protons are transferred in the rate-limiting step of ester hydrolysis while only one is transferred in peptide hydrolysis. The combination of these data with the available X-ray crystallographic, kinetic, spectroscopic, and thermodynamic data for AAP provides new insight into the catalytic mechanism of AAP.