Temperature-induced conformational transition in rabbit muscle aldolase studied by temperature dependence of sulfhydryl reactivity.
A temperature-induced non-denaturing conformational transition in rabbit muscle aldolase has been as subject of discussion and controversy for some period of time. In this study the temperature dependence of the reactivity of aldolase SH groups is investigated in order to detect subtle changes in the enzyme conformation. For model thiol-containing systems such as cysteine, glutathione and bovine serum albumin, linear Arrhenius plots have been obtained for the reaction with 5,5'-dithiobis(2-nitrobenzoic acid). On the other hand, for rabbit muscle pyruvate kinase, a protein which undergoes temperature-induced conformational transition, the plot obtained is nonlinear with a break at the temperature (18 degrees C) close to that reported earlier. In the case of aldolase the Arrhenius plots for three slowly reacting SH groups (Cys-72, 289, 338) and a fast reacting group (Cys-239) are nonlinear with a break at about 26-27 degrees C. The fluorescence measurements show that a plot of the fluorescence intensity of tryptophan residues versus temperature exhibits a break at the same temperature. It is shown that the observed conformational change is fully reversible. In the presence of the competitive inhibitor hexitol 1,6-bisphosphate, which is known to protect Cys-72 and Cys-338 from chemical modification, the Arrhenius plot exhibits a break for the fast reacting Cys-239 residue and is linear for the slowly reacting Cys-289. It is found that 0.6 M urea increases the transition temperature for all exposed SH groups of aldolase. The above results show that at several points in the aldolase molecule, including the active-site region, an abrupt change of microenvironments takes place with temperature. The competitive inhibitor protects a portion of aldolase molecule against the thermal transition.
D000818:Animals; D005634:Fructose-Bisphosphate Aldolase / Q000032:analysis*; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013696:Temperature
D013438:Sulfhydryl Compounds; D005634:Fructose-Bisphosphate Aldolase