Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.
PLoS One
; 9(9): e103902, 2014.
Article
de En
| MEDLINE
| ID: mdl-25192005
ABSTRACT
The PH1704 protease from hyperthermophilic archaean Pyrococcus horikoshii OT3 is a member of DJ-1/ThiJ/PfpI superfamily with diverse functional subclasses. The recombinant PH1704 was efficiently purified and was systematically characterized by a combination of substrate specificity analysis, steady-state kinetics study and molecular docking research. The homogeneous protease was obtained as a presumed dodecamer with molecular weight of â¼240 kDa. Iodoacetamide strongly inhibited the peptidase activity, confirming that Cys100 is a nucleophilic residue. The recombinant protein was identified as both an aminopeptidase and an endopeptidase. Experimental data showed that L-R-amc was the best substrate of PH1704. Structural interaction fingerprint analysis (SIFt) indicated the binding pose of PH1704 and showed that Tyr120 is important in substrate binding. Kinetic parameters Kcat and Kcat/Km of the Y120P mutant with L-R-amc was about 7 and 7.8 times higher than that of the wild type (WT). For the endopeptidase Y120P with AAFR-amc, Kcat and Kcat/Km is 10- and 21-fold higher than that of WT. Experimental data indicate the important functions of Tyr120 involvement in enzyme activity to form a hydrogen bond with Cys100 and as an entrance gate of the substrate with Lys43. The results of this study can be used to investigate the DJ-1/ThiJ/PfpI superfamily.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Endopeptidases
/
Pyrococcus horikoshii
Langue:
En
Journal:
PLoS One
Sujet du journal:
CIENCIA
/
MEDICINA
Année:
2014
Type de document:
Article
Pays d'affiliation:
Chine