The aminopeptidase from Aeromonas proteolytica can function as an esterase

The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester (L-Leu-OEt) with a rate of 96 +/- s(-1) and a K-m of 700 muM. 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 ESdouble dagger ester complex is 13.7 U mol(-1), while the enthalpy and entropy of activation at 25 degreesC calculated over the temperature range 298-338 K are 11.2 U mol(-1) and -175 J K-1 mol(-1), respectively. The free energy of activation at 25 degreesC 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 U 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)(H2O)/k(cat)(D2O) = 2.75 and 6.07, respectively. Proton inven tory 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.