Structure-function relationship study of a loop structure in allosteric behaviour and substrate inhibition of Lactococcus lactis prolidase
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Date
2011-02-03
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Degree Level
Masters
Abstract
Lactococcus lactis, prolidase (Llaprol) hydrolyzes Xaa-Pro dipeptides. Since Xaa-Pro is known as bitter peptides, Llaprol is potentially applicable to reduce bitterness of fermented foods. Llaprol shows allosteric behaviour and substrate inhibition, which are not reported in other prolidases. Computer models of Llaprol based on an X-ray structure of non-allosteric Pyrococcus furiosus prolidase showed that a loop structure (Loop32-43) is located at the interface of the protomers of this homodimeric metallodipeptidase. This study investigated roles of four charged residues (Asp36, His38, Glu39, and Arg40) of Loop32-43 in Llaprol using a combination of kinetic examinations of ten mutant enzymes and their molecular models. Deletion of the loop structure by Δ36-40 mutant resulted in a loss of activity, indicating Loop32-43 is crucial for the activity of Llaprol. 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 Glu39 and/or Arg40 play critical role(s) in maintaining the catalytic activity of Llaprol. These observations suggested that the loop structure is flexible and this attribute, relying on charge-charge interactions contributed by Arg40, Glu39 and Lys108, is important in maintaining the activity of Llaprol. When the loop takes a conformation close to the active site (closed state), Asp36 and His38 at the tip of the loop can be involved in the catalytic reaction of Llaprol. 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 Llaprol 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 Asp36 and Arg293 is important for the allostery of Llaprol. 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 Llaprol, implying the influence of Asp36 and His38 on the substrate binding. D36S and H38S also showed higher activities at pH 5.0 to 6.0, in which range WT Llaprol steeply decreased its activity, indicating Asp36 and His38 are involved in the active centre and influence the microenvironment of catalytic His296. 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 Llaprol mutants.
From these results, we concluded that the unique behaviours of Llaprol are correlated to Loop32-43 and Asp36 and His38 on it. When Loop32-43 takes a closed conformation, Asp36 interacts with Arg293 via charge-charge attraction to form an allosteric subsite. The saturation of the allosteric site with substrates further allowed the communications of His38 with S1 site residues to complete the active site. When the substrate concentration becomes higher than it is required to saturated productive S1' site, His38, Phe190 and Arg293 would resemble the residue arrangement of S1' site residues (His292, Tyr329, and Arg337) and bind to the proline residue of substrates. This non-productive binding would prevent the conformational change of Loop32-43, 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 Llaprol proteins.
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Keywords
Site-directed mutagenesis, Allosteric subsite, X-ray diffraction
Citation
Degree
Master of Science (M.Sc.)
Department
Applied Microbiology and Food Science
Program
Applied Microbiology and Food Science