Naslov
Absolutely conserved tryptophan in M49 family of peptidases contributes to catalysis and binding of competitive inhibitors

Autori
Špoljarić, Jasminka ; Salopek-Sondi, Branka ; Makarević, Janja ; Vukelić, Bojana ; Agić, Dejan ; Šimaga, Šumski ; Jajčanin-Jozić, Nina ; Abramić, Marija

Izvornik
Bioorganic chemistry (0045-2068) 37 (2009), 1-3; 70-76

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
dipeptidyl peptidase III ; hydroxamate inhibitor ; peptidase family M49 ; protein structure-function ; site-directed mutagenesis

Sažetak
The role of the unique fully conserved tryptophan in metallopeptidase family M49 (dipeptidyl peptidase III family) was investigated by site-directed mutagenesis on human dipeptidyl peptidase III (DPP III) where Trp300 was subjected to two substitutions (W300F and W300L). The mutant enzymes showed thermal stability equal to the wild-type DPP III. Conservative substitution of the Trp300 with phenylalanine decreased enzyme activity 2-4 – fold, but did not significantly change the Km values for two dipeptidyl 2-naphthylamide substrates. However, the Km for the W300L mutant was elevated five-fold and the kcat value was reduced 16-fold with Arg-Arg-2-naphthylamide. Both substitutions had a negative effect on the binding of two competitive inhibitors designed to interact with S1 and S2 subsites. These results indicate the importance of the aromatic nature of W300 in DPP III ligand binding and catalysis, and contribution of this residue in maintaining the functional integrity of this enzyme's S2 subsite.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA