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Structure-function relationship study of a loop structure in allosteric behaviour and substrate inhibition of <i>Lactococcus lactis</i> prolidase

<p><i>Lactococcus lactis,</i> prolidase (<i>Lla</i>prol) hydrolyzes Xaa-Pro dipeptides. Since Xaa-Pro is known as bitter peptides, <i>Lla</i>prol is potentially applicable to reduce bitterness of fermented foods. <i>Lla</i>prol shows allosteric behaviour and substrate inhibition, which are not reported in other prolidases. Computer models of <i>Lla</i>prol based on an X-ray structure of non-allosteric <i>Pyrococcus furiosus</i> prolidase showed that a loop structure (Loop<sup>32-43</sup>) is located at the interface of the protomers of this homodimeric metallodipeptidase. This study investigated roles of four charged residues (Asp<sup>36</sup>, His<sup>38</sup>, Glu<sup>39</sup>, and Arg<sup>40</sup>) of Loop<sup>32-43</sup> in <i>Lla</i>prol using a combination of kinetic examinations of ten mutant enzymes and their molecular models. Deletion of the loop structure by Î36-40 mutant resulted in a loss of activity, indicating Loop<sup>32-43</sup> is crucial for the activity of <i>Lla</i>prol. D36S and H38S exhibited 96.2 % and 10.3 % activity of WT, whereas little activities (less than 1.0 % of WT activity) were observed for mutants E39S, D36S/E39S, R40S, R40E, R40K and H38S/R40S. These results implied that Glu<sup>39</sup> and/or Arg<sup>40</sup> play critical role(s) in maintaining the catalytic activity of <i>Lla</i>prol. These observations suggested that the loop structure is flexible and this attribute, relying on charge-charge interactions contributed by Arg<sup>40</sup>, Glu<sup>39</sup> and Lys<sup>108</sup>, is important in maintaining the activity of <i>Lla</i>prol. When the loop takes a conformation close to the active site (closed state), Asp<sup>36</sup> and His<sup>38</sup> at the tip of the loop can be involved in the catalytic reaction of <i>Lla</i>prol. The two active mutant prolidases (D36S and H38S) resulted in modifications of the unique characteristics; the allosteric behaviour was not observed for D36S, and H38S <i>Lla</i>prol showed no substrate inhibition. D36E/R293K, maintaining the negative charge of position 36 and positive charge of position 293, still possessed the allosteric behaviour, whereas the loss of the charges at these positions (D36S of this study and R293S of a previous study (Zhang et al., 2009 BBA-Proteins Proteom 1794, 968-975) eliminated the allosteric behaviour. These results indicated the charge-charge attraction between Asp<sup>36</sup> and Arg<sup>293</sup> is important for the allostery of <i>Lla</i>prol. In the presence of either zinc or manganese divalent cations as the metal catalytic centre, D36S and H38S enzymes also showed different substrate preferences from WT <i>Lla</i>prol, implying the influence of Asp<sup>36</sup> and His<sup>38</sup> on the substrate binding. D36S and H38S also showed higher activities at pH 5.0 to 6.0, in which range WT <i>Lla</i>prol steeply decreased its activity, indicating Asp<sup>36</sup> and His<sup>38</sup> are involved in the active centre and influence the microenvironment of catalytic His<sup>296</sup>. The above observations are attributed to modifications of their local structure in the active centre since the temperature dependency and thermal denaturing temperature indicated little effects on the overall structure of the <i>Lla</i>prol mutants.</p>
<p>From these results, we concluded that the unique behaviours of <i>Lla</i>prol are correlated to Loop<sup>32-43</sup> and Asp<sup>36</sup> and His<sup>38</sup> on it. When Loop<sup>32-43</sup> takes a closed conformation, Asp<sup>36</sup> interacts with Arg<sup>293</sup> via charge-charge attraction to form an allosteric subsite. The saturation of the allosteric site with substrates further allowed the communications of His<sup>38</sup> with S<sub>1</sub> site residues to complete the active site. When the substrate concentration becomes higher than it is required to saturated productive S<sub>1</sub>' site, His<sup>38</sup>, Phe<sup>190</sup> and Arg<sup>293</sup> would resemble the residue arrangement of S<sub>1</sub>' site residues (His<sup>292</sup>, Tyr<sup>329</sup>, and Arg<sup>337</sup>) and bind to the proline residue of substrates. This non-productive binding would prevent the conformational change of Loop<sup>32-43</sup>, which further results in the substrate inhibition. For further confirmation of this mechanism, crystallographic studies will be conducted. In this thesis, we have indentified the conditions to produce crystals of <i>Lla</i>prol proteins.</p>

Identiferoai:union.ndltd.org:USASK/oai:usask.ca:etd-02252011-095142
Date25 February 2011
CreatorsChen, Jian An
ContributorsPawel Grochulski, Takuji Tanaka, George Khachatourians, Louis Delbaere, Vladimir Vujanovic
PublisherUniversity of Saskatchewan
Source SetsUniversity of Saskatchewan Library
LanguageEnglish
Detected LanguageEnglish
Typetext
Formatapplication/pdf
Sourcehttp://library.usask.ca/theses/available/etd-02252011-095142/
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