Szeltner_2009_Biochim.Biophys.Acta_1794_1204

We have overexpressed in E. coli, purified and investigated the kinetic, thermodynamic and biophysical properties of an acylaminoacyl peptidase (AAP), from the thermophile Pyrococcus horikoshii (PhAAP). It was shown that the electrostatic environment of the catalytic site of PhAAP substantially influenced the pH dependence of the specificity rate constant (k(cat)/K(m)). However, 0.3 M NaCl, which depressed the electrostatic effects, simplified the complex pH-rate profile. The rate of formation of the enzyme-substrate complex (k(1)) was obtained from a non-linear Arrhenius plot. The lack of substrate leaving group effects indicated that k(1) is the rate determining step in the catalysis. DSC and CD measurements demonstrated that PhAAP displayed a stable structure in the catalytically competent pH range. It was shown that PhAAP is not just an acylaminoacyl peptidase, but it also has an endopeptidase activity and so differs from the mammalian AAPs. Size exclusion chromatography with PhAAP revealed a hexameric structure, which is unique among the known members of the prolyl oligopeptidase family that includes AAPs and suggests that its cellular function may be different from that of the dimeric AAP also found in the same organism.