Title

The Aminopeptidase from Aeromonas proteolytica can Function as an Esterase

Document Type

Article

Publication Date

1-2002

Source Publication

Journal of Biological Inorganic Chemistry

Source ISSN

0949-8257

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, kH2Ocat/kD2Ocat=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.

Comments

Journal of Biological Inorganic Chemistry, Vol. 7, No. 1-2 (January 2002): 129-135. DOI.

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