Substrate Report for: Ac-Leu-p-nitroanilide

Acylaminoacylpeptidase (AAP) belongs to peptidase protein family (POP), which can degrade amyloid beta-peptide forms in the brains of patients and hence leads to Alzheimer's disease. And so, AAP is considered to be a novel target in the design of drugs against Alzheimer's disease. In this investigation, six molecular dynamics simulations were used to find that the interaction between the wild-type and R526V AAP with two different substrates (p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide). Our results were as follows: firstly, Ac-Leu-p-nitroanilide bound to R526V AAP to form a more disordered loop (residues 552-562) in the alpha/beta-hydrolase fold like of AAP, which caused an open and inactive AAP domain form, secondly, binding p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide to AAP can decrease the flexibility of residues 225-250, 260-270 and 425-450, in which the ordered secondary structures may contain the suitable geometrical structure and so it is useful to serine attack. Our theoretical results showed that the binding of the two substrates can induce specific conformational changes responsible for the diverse AAP catalytic specificity. These theoretical substrate-induced structural diversities can help explain the abilities of AAPs to recognize and hydrolyze extremely different substrates.

Acylpeptide hydrolases (APH) catalyze the removal of an N-acylated amino acid from blocked peptides. APH is significantly more sensitive than acetylcholinesterase, a target of Alzheimer's disease, to inhibition by organophosphorus (OP) compounds. Thus, OP compounds can be used as a tool to probe the physiological functions of APH. Here, we report the results of a computational study of molecular dynamics simulations of APH bound to the OP compounds and an exploration of the chlorpyrifos escape pathway using steered molecular dynamics (SMD) simulations. In addition, we apply SMD simulations to identify potential escape routes of chlorpyrifos from hydrolase hydrophobic cavities in the APH-inhibitor complex. Two previously proposed APH pathways were reliably identified by CAVER 3.0, with the estimated relative importance of P1 > P2 for its size. We identify the major pathway, P2, using SMD simulations, and Arg526, Glu88, Gly86, and Asn65 are identified as important residues for the ligand leaving via P2. These results may help in the design of APH-targeting drugs with improved efficacy, as well as in understanding APH selectivity of the inhibitor binding in the prolyl oligopeptidase family.

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.