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Characterization of a novel acylaminoacyl peptidase with hexameric structure and endopeptidase activity

Szeltner, Zoltán

Kiss, András

Domokos, Klarissza

Harmat, Veronika

Náray-Szabó, Gábor

Polgár, László

Elsevier

2009

QD05 Crystallography / kristálytan

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 (kcat/Km). 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 (k1) was obtained from a non-linear Arrhenius plot. The lack of substrate leaving group effects
indicated that k1 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.

Article

PeerReviewed

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Szeltner, Zoltán and Kiss, András and Domokos, Klarissza and Harmat, Veronika and Náray-Szabó, Gábor and Polgár, László (2009) Characterization of a novel acylaminoacyl peptidase with hexameric structure and endopeptidase activity. Biochimica et Biophysica Acta, 1794 (8). pp. 1204-1210. ISSN 1570-9639

http://www.sciencedirect.com/science/article/pii/S157096390...

http://real.mtak.hu/2821/