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 +/- 5 s-1 and a Km of 700 microM. 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 ( Ea) for the activated ES ester complex is 13.7 kJ mol-1, while the enthalpy and entropy of activation at 25 degrees 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 degrees 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, 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.
Bienvenue
David L
DL
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA.
Mathew
Rebecca S
RS
Ringe
Dagmar
D
Holz
Richard C
RC
D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D004795:Enzyme Stability / Q000502:physiology; D004950:Esterases / Q000737:chemistry / Q000378:metabolism*; D004952:Esters / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013696:Temperature; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism*
D001426:Bacterial Proteins; D004952:Esters; D006639:Histidine; D004950:Esterases; D000626:Aminopeptidases; C038222:bacterial leucyl aminopeptidase; D015032:Zinc