Glu88 in the non-catalytic domain of acylpeptide hydrolase plays dual roles: Charge neutralization for enzymatic activity and formation of salt bridge for thermodynamic stability

Abstract

Acylpeptide hydrolase of Aeropyrum pernix K1 is composed of a catalytic α/β hydrolase domain and a non-catalytic β-propeller domain. The Glu88 residue of the propeller domain is highly conserved in the prolyl oligopeptidase family and forms an inter-domain salt bridge with Arg526, a key residue for substrate binding. We have dissected the functions of Glu88 using site-directed mutagenesis, steady-state kinetics analyses, and molecular dynamics simulations. In E88A and E88A/R526K mutants, with a broken inter-domain salt bridge and a positive charge at position 526, catalytic activities for both a peptidase substrate and an esterase substrate were almost abolished. Analysis of the pH dependence of the mutants' reaction kinetics indicates that these mutations lead to changes in the electrostatic environment of the active site, which can be modulated by chloride ions. These findings indicate that the neutralization at position 526 is favorable for the activity of the enzyme, which is also verified by the catalytic behavior of E88A/R526V mutant. All mutants have lower thermodynamic stability than the wild-type. Therefore, Glu88 plays two major roles in the function of the enzyme: neutralizing the positive charge of Arg526, thereby increasing the enzymatic activity, and forming the Glu88–Arg526 salt bridge, thereby stabilizing the protein.

Introduction

Acylpeptide hydrolases (APH), which catalyze the removal of an N-acylated amino acid from blocked peptides [1], [2], are widely distributed in archaea, bacteria, and eucarya [3], [4], [5], [6]. APH is a member of the prolyl oligopeptidase (POP) family, an extensively investigated class of serine proteases as potential pharmaceutical targets for neurological diseases [7], [8], [9]. Compared with the classic serine proteases such as trypsin, chymotrypsin and subtilisin, the POP family exhibits many differences in structural features and catalytic behavior. For example, members of the POP family consist of two domains, an α/β hydrolase domain and a β-propeller domain, while classic serine proteases have radically different β/β (chymotrypsin) and α/α (subtilisin) protein scaffolds, although they have similar catalytic triads [10], [11], [12]. The activity of the POP family is also very sensitive to the ionic strength of its environment, whereas trypsin, chymotrypsin, and subtilisin are not [13], [14], [15], [16]. Although structural similarity is quite high in POP family members, their sequence homology is very low due to a long period of divergent evolution [17]. Consequently, interactions between conserved residues may play important roles in POP function and structure.

The APH gene (APE1547) from the thermophilic archaeon Aeropyrum pernix K1 (termed apAPH) has been cloned and over-expressed in Escherichia coli [18]. The recombinant enzyme has an optimal temperature at 90 °C. It is extremely stable and it shows hydrolytic activity towards a wide range of substrates, including p-nitrophenyl alkanoate esters of varying alkyl chain lengths, pNA-labeled amino acids, and peptides [18]. In a previous study we determined the crystal structure of apAPH complexed with an organo-phosphorus substrate [19]. apAPH exhibits a canonical POP family structure: the N-terminal domain is a regular seven-bladed β-propeller while the C-terminal domain has an α/β hydrolase fold which includes the active site and a conserved Ser445–Asp524–His556 catalytic triad. The active site is located in the inter-domain region. The domain interface is stabilized by 29 hydrogen bonds and salt bridges, with additional stability provided by hydrophobic forces [19]. Recently, we found that the conserved Arg526 of apAPH in the active site plays a crucial role in the discrimination of the esterase and peptidase activities [20]. Structural analysis revealed that a conserved residue, Glu88, in the noncatalytic domain forms an inter-domain salt bridge with Arg526 (Fig. 1). This inter-domain salt bridge is highly conserved among the POP family. In general, it links the Arg at the S2 substrate binding pocket in the α/β hydrolase domain and a Glu/Asp on the second blade of the β-propeller domain. The functions of these two conserved residues have already attracted some attention, and it has been shown that even conserved mutageneses at either corresponding residue in Myxococcus xanthus POP can result in > 99% losses of activity with peptide substrates [21]. Similarly, mutations at the corresponding residue of Glu88 in human dipeptidyl-peptidase IV have reduced the activity more than ten-fold [22]. Abbott et al. proposed an explanation for these observations that it is involved tight anchoring of the substrate by the Glu88–Arg526 salt bridge. However, the functions of Glu88 as well as the reason of the high conservation of the salt bridge were not studied in detail.

In the investigations reported here, the functions of Glu88 and the salt bridge of the inter-domain were systemically studied by site-directed mutagenesis and kinetic analysis. Further insight into the mechanism of activity regulation was gained by examining the effects of pH and ionic strength on enzyme kinetics. Finally, molecular dynamics (MD) simulations were undertaken with selected apAPH mutants to elucidate the structural importance of the inter-domain interaction. Our findings show that the Glu88 residue in the non-catalytic propeller domain regulates the catalytic domain, via an inter-domain interaction, in a manner that has not been previously described.