| Protease is a group of enzymes, which can catalyze the hydrolysis of peptide linkage ofpolypeptide chain and plays an important role in biological function. Compared withmesophilic proteases, the proteases from hyperthermophiles exhibited high thermostability,and thus the study on their thermal stability and catalytic mechanism has been a hot topic inenzymology. The PhpI protease from hyperthermophilic archaeon Pyrococcus horikoshiiOT3 is a member of PfpI peptidase family, which belongs to the DJ-1 superfamily. TheDJ-1 superfamily members are widely distributed in nearly all the tissues, and have similar3D structure and diverse functions, including protease, chaperone protein, kinase, kinase,and thiamine synthesis of transcription regulatory elements. Herein, computational biologyand protein engineering methods were employed to understand the reaction mechanism andthe related functional sites.In the previous work, the PhpI protease gene were successfully cloned, over-expressed in E.coli, and characterized with respect to substrate specificity and catalytic mechanism. Theoptimal temperature and pH were 80 oC and 8.0, respectively. In the present study, theprotease was purified to a single band of the target protein using the combination ofthermal denaturation and gel filtration chromatography, and the active form wascharacterized of a dodecamer. Activity analysis showed that besides the high activitytowards natural substrate gelatin, PhpI exhibited the aminopeptidase and endopeptidaseactivity. The highest activity was obtained with the aminopeptidase substrate L-R-AMC,about 10 times higher than endopeptidase substrate L-AAFR-AMC. The kinetic parameterskcat, Km and kcat/Km of aminopeptidase were 0.6348 min-1, 12μM and 0.052μM-1 min-1,respectively; the corresponding values of endopeptidase were 0.112 min-1, 10μM and0.011μM-1min-1, respectively. No matter for aminopeptidase or endopeptidase activity,protease PhpI preferred arginine on the P1 site. Through the crystal structure and substrate docking analysis, we found that the benzenering of Tyr120 at the entrance of substrate binding pocket might hinder the access ofsubstrate molecules. Meanwhile, the hydrogen bond between N atom of the Y120 mainchain and O atom of nucleophilic residue Cys100 might also played a key role in thecatalytic efficiency. Therefore, Tyr120 was selected as the mutation site for furtherinvestigation, and the mutants Y120S, Y120W and Y120P were designed to explore theeffects of different side chain residues and substrate binding pocket on the enzyme catalyticactivity. Different mutants directly changed the diameter of substrate binding pocket, andthus significantly affected their catalytic activity. Particularly, the kcat/Km values of Y120Pfor the aminopeptidase and endopeptidase substrate were about 7 times and 20 times higherthan the wild type, respectively. Molecular docking showed that the benzene ring of Tyr120side chain increased the steric hindrance of substrate binding pocket and the hydrogen bondbetween Cys100 and Tyr120 hindered the binding of substrate molecule, which were bothunfavorable for the catalytic efficiency. Thus, Tyr120 was determined as a key site in thesubstrate binding and catalytic process.Structural analysis showed that an allosteric regulation might be present in the proteasePhpI due to its special polymeric structure. The preliminary kinetic data indicated that allthe Hill coefficients for different substrates were less than 1, which approved of a negativesynergistic effect. A sulfate radical SO42- located in the interface of two adjacent subunits,and formed two salt bridges between Arg113, and also formed hydrogen bond with Asn129through water molecule. In addition, Tyr120 were also located in the same loop region withArg113 and Asn129. Therefore, it was concluded that Arg113 and Asn129 might be theallosteric factors, and had a significant impact on the catalytic activity.Based on the above analysis, the mutants R113A, R113T, N129A and N129D wereconstructed, and the kinetic parameters were determined using the substrates L-R-AMCand L-AAFR-AMC. The activity of R113A and R113T was remarkably improved, whilethe activity of N129A and N129D was decreased, even no detectable activity on thesubstrate L-AAFR-AMC for N129D. It was presumed that the mutation sites 113 and 129located in the same loop region with Tyr120, and thus affected the catalytic activity by changing the conformation of the entire loop region.For the mutant N129D, due to the loss of SO42-, the helix which site 129 located on wouldchange into a loop region. In the catalytic triad, His101 and Glu474 were from differentsubunits, and the hydrogen bonding interaction was generated in the interface between twosubunits. The stronger the effect of hydrogen bonds, cysteine nucleophile polarization wasstronger, the more prone to catalytic reactions. Once the enzyme lost its anions, thehydrogen bond between His101 and Glu474 would be intensively disturbed and furtheraffected the catalytic activity, which would be an explanation for the loss of catalyticactivity of N129D. The distances form 113 and 129 sites to the active site were more than10?, which provided a new insight for remote regulation and control.In this study, we determined some important residues for the catalytic activity of proteasePhpI, and all the residues had their own effects on the catalytic activity. The work wouldprovide an important foundation for the exploration of catalytic mechanism of proteasePhpI.
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