Limits...
The Thermal Stability of the Fusarium solani pisi Cutinase as a Function of pH.

Petersen SB, Fojan P, Petersen EI, Petersen MT - J. Biomed. Biotechnol. (2001)

Bottom Line: The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour.The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA.We propose a molecular interpretation for the pH-variation in enzymatic activity.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
We have investigated the thermal stability of the Fusarium solani pisi cutinase as a function of pH, in the range from pH 2-12. Its highest enzymatic activity coincides with the pH-range at which it displays its highest thermal stability. The unfolding of the enzyme as a function of pH was investigated by microcalorimetry. The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour. The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA. We propose a molecular interpretation for the pH-variation in enzymatic activity.

No MeSH data available.


Titration curve of cutinase predicted by TITRA Plot of net charge of cutinase versus pH, calculated at a step width of 0.5 pH units.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC113781&req=5

Figure 5: Titration curve of cutinase predicted by TITRA Plot of net charge of cutinase versus pH, calculated at a step width of 0.5 pH units.

Mentions: The different apparent onset of enzymatic activity compared to the thermal stability profile as a function of pH can be explained by the different titration states of the active site residues (Figure 5). The formation of an active enzyme includes the correct formation of a hydrogen bond network in and around the active site, not only involving the residues of the catalytic triad, but also Tyr119, neighbouring the active site Ser120. This Tyrosine residue forms with its hydroxyl group a hydrogen bond with the backbone oxygen of the catalytic His188. The titration state of the catalytic triad (SER120, HIS188, and ASP175) is responsible for the hydrogen bonding among these three residues and for the level of enzymatic activity. The core residuein the triad is HIS188. The hydrogen network in the catalytic triad is established when HIS188 becomes deprotonated. Histidines usually display pK values between pH 6 to 7. The electrostatic potential of the active site environment is shown as a GRASP figure (Figure 6). At the pH withhighest enzymatic activity the surrounding of the active site shows a slight negative electrostatic potential. This negative potential is achieved mainly due to the deprotonation of ASP175, GLU44 and deprotonation of the catalytic His188. Furthermore, the active site surrounding is populated with a set of tyrosine residues, contributing to the overall negative electrostatic potential at basic pH values. The lower Tm observed below pH 4.0 is likely due to the predominance of repulsive electrostatic forces, since at very acidic pH values the protein carries an excess of positive charge (see potential distribution at pH 4.0). This excess positive charge might also prevent the unfavourable hydrophobic interactions leading to irreversible aggregation in the neutral to basic pH region. This excess of positive charge will only be compensatedfor after deprotonation of Aspartic and Glutamic acid residues (pKa approx. 4–4.5). This coincides also with the rise in Tm values of cutinase after pH 4.5. The predicted pK values for E and D are close to the initial values for theseamino acids, with the exception for D22 (pK 2.72) and E60 (pK 2.95), which are found to form a salt bridge with R20 (pK 12.94) and E131 (pK 3.72) and D132 (pK 3.14) are linked in another saltbridge with R88 (pK 12.98) on the surface of cutinase (Figure 7). This explains also their drastically shifted pK values. At maximum enzymatic activity (around pH 8.5) Aspartic and Glutamic acid residues are negatively charged, His188 of the catalytic triad ismost likely neutral, tyrosine residues are predominantly protonated and Lysine and Arginine are positively charged. The electrostatic potential surrounding the active site is slightly negative (Figure 6). After pH 9–10 a drop in both Tm and catalytic activity is observed. This coincides with the titration of Tyrosine residues (pK values predicted by TITRA between 9.8–12.4, at pH 8.5). The excess of negative electrostatic potential in the active site due to deprotonation of Tyrosine residues can be seen in Figure 6. The Tyrosine residues and here especially Tyr119, once deprotonated loose their capability of donating hydrogens to hydrogen bonds and at the same time add to the molecule surface an excess of negative charge that is likely to destabilize the 3D structure of cutinase. This excess of negative charge in theactive site and close to the catalytic triad may lead to a considerable structural destabilisation of the catalytic triad, since it is also in close proximity to the catalytic ASP175. The strong repulsion forces lead to a dramatic decrease not only in enzymatic activity, but also leads to a thermal destabilisation. This is in agreement with lower enzymatic activity as well as lowered thermal stability at very basic pH values.


The Thermal Stability of the Fusarium solani pisi Cutinase as a Function of pH.

Petersen SB, Fojan P, Petersen EI, Petersen MT - J. Biomed. Biotechnol. (2001)

Titration curve of cutinase predicted by TITRA Plot of net charge of cutinase versus pH, calculated at a step width of 0.5 pH units.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113781&req=5

Figure 5: Titration curve of cutinase predicted by TITRA Plot of net charge of cutinase versus pH, calculated at a step width of 0.5 pH units.
Mentions: The different apparent onset of enzymatic activity compared to the thermal stability profile as a function of pH can be explained by the different titration states of the active site residues (Figure 5). The formation of an active enzyme includes the correct formation of a hydrogen bond network in and around the active site, not only involving the residues of the catalytic triad, but also Tyr119, neighbouring the active site Ser120. This Tyrosine residue forms with its hydroxyl group a hydrogen bond with the backbone oxygen of the catalytic His188. The titration state of the catalytic triad (SER120, HIS188, and ASP175) is responsible for the hydrogen bonding among these three residues and for the level of enzymatic activity. The core residuein the triad is HIS188. The hydrogen network in the catalytic triad is established when HIS188 becomes deprotonated. Histidines usually display pK values between pH 6 to 7. The electrostatic potential of the active site environment is shown as a GRASP figure (Figure 6). At the pH withhighest enzymatic activity the surrounding of the active site shows a slight negative electrostatic potential. This negative potential is achieved mainly due to the deprotonation of ASP175, GLU44 and deprotonation of the catalytic His188. Furthermore, the active site surrounding is populated with a set of tyrosine residues, contributing to the overall negative electrostatic potential at basic pH values. The lower Tm observed below pH 4.0 is likely due to the predominance of repulsive electrostatic forces, since at very acidic pH values the protein carries an excess of positive charge (see potential distribution at pH 4.0). This excess positive charge might also prevent the unfavourable hydrophobic interactions leading to irreversible aggregation in the neutral to basic pH region. This excess of positive charge will only be compensatedfor after deprotonation of Aspartic and Glutamic acid residues (pKa approx. 4–4.5). This coincides also with the rise in Tm values of cutinase after pH 4.5. The predicted pK values for E and D are close to the initial values for theseamino acids, with the exception for D22 (pK 2.72) and E60 (pK 2.95), which are found to form a salt bridge with R20 (pK 12.94) and E131 (pK 3.72) and D132 (pK 3.14) are linked in another saltbridge with R88 (pK 12.98) on the surface of cutinase (Figure 7). This explains also their drastically shifted pK values. At maximum enzymatic activity (around pH 8.5) Aspartic and Glutamic acid residues are negatively charged, His188 of the catalytic triad ismost likely neutral, tyrosine residues are predominantly protonated and Lysine and Arginine are positively charged. The electrostatic potential surrounding the active site is slightly negative (Figure 6). After pH 9–10 a drop in both Tm and catalytic activity is observed. This coincides with the titration of Tyrosine residues (pK values predicted by TITRA between 9.8–12.4, at pH 8.5). The excess of negative electrostatic potential in the active site due to deprotonation of Tyrosine residues can be seen in Figure 6. The Tyrosine residues and here especially Tyr119, once deprotonated loose their capability of donating hydrogens to hydrogen bonds and at the same time add to the molecule surface an excess of negative charge that is likely to destabilize the 3D structure of cutinase. This excess of negative charge in theactive site and close to the catalytic triad may lead to a considerable structural destabilisation of the catalytic triad, since it is also in close proximity to the catalytic ASP175. The strong repulsion forces lead to a dramatic decrease not only in enzymatic activity, but also leads to a thermal destabilisation. This is in agreement with lower enzymatic activity as well as lowered thermal stability at very basic pH values.

Bottom Line: The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour.The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA.We propose a molecular interpretation for the pH-variation in enzymatic activity.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
We have investigated the thermal stability of the Fusarium solani pisi cutinase as a function of pH, in the range from pH 2-12. Its highest enzymatic activity coincides with the pH-range at which it displays its highest thermal stability. The unfolding of the enzyme as a function of pH was investigated by microcalorimetry. The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour. The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA. We propose a molecular interpretation for the pH-variation in enzymatic activity.

No MeSH data available.