Significance of the Conserved Tyr352 and Asp380 Residues in the Catalytic Activity of Bacillus stearothermophilus Aminopeptidase II as Evaluated by Site-directed Mutagenesis

Abstract

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.

Aminopeptidase T of M29 Family Acts as A Novel Intracellular Virulence Factor for Listeria monocytogenes Infection

“…Structural information on clan MQ peptidases has so far relied exclusively on the crystal structures of AmpS 13 and AmpT 17 . In addition, most of the previous studies on peptidases in the M29 family focused on their biochemical properties and biophysical characterizations 18 19 20 . Despite the emerging roles of aminopeptidases, the members in the M29 family remain poorly understood.…”

Biophysical characterization of a recombinant aminopeptidase II from the thermophilic bacterium Bacillus stearothermophilus

“…The recombinant enzyme exhibited a marked preference for leucinep-nitroanilide (Leup-NA) and was sensitive to oxidative damage by hydrogen peroxide [19]. Site-directed mutagenesis was further conducted to identify residues essential for the catalytic activity of His 6 -tagged BsAmpII [20][21][22]. Although some researchers have focused their studies on the biochemical and structural characterization of clan MQ peptidases, the biophysical properties of this group of enzymes have not been explored before.…”