Pressure and Denaturants in the Unfolding of Triosephosphate Isomerase: The Monomeric Intermediates of the Enzymes from Saccharomyces cerevisiae and Entamoeba histolytica

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• 作者：Adri&aacute ; n R. V&aacute ; zquez-Pé ; rez ; D. Alejandro Fern&aacute ; ndez-Velasco
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：July 24, 2007
• 年：2007
• 卷：46
• 期：29
• 页码：8624 - 8633
• 全文大小：141K
• 年卷期：v.46,no.29(July 24, 2007)
• ISSN：1520-4995

文摘

Triosephosphate isomerase (TIM) is a dimeric enzyme formed by two identical (images/gifchars/beta2.gif" BORDER=0 ALIGN="middle">/images/gifchars/alpha.gif" BORDER=0>)₈ barrels.In this work, we compare the unfolding and refolding of the TIMs from Entamoeba histolytica (EhTIM)and baker's yeast (yTIM). A monomeric intermediate was detected in the GdnHCl-induced unfolding ofEhTIM. The thermodynamic, spectroscopic, catalytic, and hydrodynamic properties of this intermediatewere found to be very similar to those previously described for a monomeric intermediate of yTIM observedin GdnHCl. Monomer unfolding was reversible for both TIMs; however, the dissociation step was reversiblein yTIM and irreversible in EhTIM. Monomer unfolding induced by high pressure in the presence ofGdnHCl was a reversible process. images/gifchars/Delta.gif" BORDER=0 >G_Unf, images/gifchars/Delta.gif" BORDER=0 >V_Unf, and P_1/2 were obtained for the 0.7-1.2 M GdnHClrange. The linear extrapolation of these thermodynamic parameters to the absence of denaturant showedthe same values for both intermediates. The images/gifchars/Delta.gif" BORDER=0 >V_UnfH2O values calculated for EhTIM and yTIM monomericintermediates are the same within experimental error (-57 ± 10 and -76 ± 14 mL/mol, respectively).These images/gifchars/Delta.gif" BORDER=0 >V_{Unf H2O} values are smaller than those reported for the unfolding of monomeric proteins of similarsize, suggesting that TIM intermediates are only partially hydrated. images/entities/verbar.gif">images/gifchars/Delta.gif" BORDER=0 >V_Unfimages/entities/verbar.gif"> increased with denaturantconcentration; this behavior is probably related to structural changes in the unfolded state induced byGdnHCl and pressure. From the thermodynamic parameters that were obtained, it is predicted that in theabsence of denaturants, pressure would induce monomer unfolding (P_1/2 ~ 140 MPa) prior to dimerdissociation (P_1/2 ~ 580 MPa). Therefore, dimerization prevents the pressure unfolding of the monomer.