[Show abstract][Hide abstract] 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 (Arg526) 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 ∼7 times higher than the peptidase activity with Ac-Leu-p-nitroanilide as substrate. However, with the same substrates, this difference was increased to ∼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 kcat and Km of the enzyme were discussed based on molecular dynamics simulation studies.
[Show abstract][Hide abstract] ABSTRACT: To enhance the enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1 (APE1547), a directed evolution approach is employed to generate mutant library from the native enzyme. A mutation (TBC26) is identified after one round of epPCR. The enantioselectivity of TBC26 is increased up to 2.6-fold compared to that of wild type enzyme. TBC26 contains five amino acid substitutions (R11G, L36P, V223A, I551L, A564T). The five mutation sites are spatially distant to the catalytic center. According to the published crystal structure of WT and considering the changes of secondary and tertiary structure, here we try to explain the change of enantioselectivity of the TBC26. The results suggest that the change of enantioselectivity of enzyme has a close relationship to the configuration of the enzyme.
Journal of Molecular Catalysis B Enzymatic 03/2006; 38(3):148-153. DOI:10.1016/j.molcatb.2005.11.008 · 2.13 Impact Factor