Discrimination of Esterase and Peptidase Activities of Acylaminoacyl Peptidase from Hyperthermophilic Aeropyrum pernix K1 by a Single Mutation

Qiuyan Wang,Guangyu Yang,Yanli Liu,Yan Feng

doi:10.1074/jbc.m601015200

Abstract

It has been shown that highly conserved residues that form crucial structural elements of the catalytic apparatus may be used to account for the evolutionary history of enzymes. Using saturation mutagenesis, we investigated the role of a conserved residue (Arg(526)) at the active site of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 in substrate discrimination and catalytic mechanism. This enzyme has both peptidase and esterase activities. The esterase activity of the wild-type enzyme with p-nitrophenyl caprylate as substrate is approximately 7 times higher than the peptidase activity with Ac-Leu-p-nitroanilide as substrate. However, with the same substrates, this difference was increased to approximately 150-fold for mutant R526V. A more dramatic effect occurred with mutant R526E, which essentially completely abolished the peptidase activity but decreased the esterase activity only by a factor of 2, leading to a 785-fold difference in the enzyme activities. These results provide rare examples that illustrate how enzymes can be evolved to discriminate their substrates by a single mutation. The possible structural and energetic effects of the mutations on k(cat) and K(m) of the enzyme were discussed based on molecular dynamics simulation studies.

Highlights

Nificant sequence homology between lipases and peptidases, except for a 10-residue segment near the catalytic Ser
It has been suggested that the POP family has an evolutionary relationship with the microbial lipase family, little work has been done to determine the relationship between peptidase and esterase activity of the POP family
We performed saturation mutagenesis at the potential substrate binding residue Arg526 in apAPH and found that it is an important residue for the substrate specificity between peptidase and esterase

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strain, and Plasmid—Restriction enzyme DpnI was purchased from Promega (Madison, WI). A 20-␮l aliquot from each well was pipetted into a new 96-well plate, to which 200 ␮l of substrate solution containing 0.2 mM Ac-LeupNA or pNPC8 in 50 mM phosphate buffer at pH 8.0 was added. The esterase activity of apAPH was measured by the release of p-nitrophenyl from pNPC8, which was released in a way similar to that described for the hydrolysis of Ac-Leu-pNA. Measurement of Steady-state Kinetics—The kinetic parameters Km and kcat of wild type and the mutants were determined at 80 °C for Ac-Leu-pNA and pNPC8. Determination of Individual Rate Constants and Activation Energies for Hydrolysis of pNPC8 and Ac-Leu-pNA—Individual rate constants defining the mechanism of substrate hydrolysis were extracted from the values of kcat/Km obtained as a function of temperature from 40 to 90 °C in 50 mM phosphate buffer at pH 8.0. Because the Michaelis-Menten parameters s and kcat are composite functions of the individual kinetic rates in Scheme 1, measurements of s and kcat as a function of temperature can resolve all of the parameters from Equations 3 and 4 (16 –19),

11 T Ϫ T0 ϩ k3 exp ϪER3

RESULTS

DISCUSSION