title: Reversible conformational transition gives rise to 'zig-zag' temperature dependence of the rate constant of irreversible thermoinactivation of enzymes.
abstract: We have obtained unusual 'zig-zag' temperature dependencies of the rate constant of irreversible thermoinactivation (k(in)) of enzymes (alpha-chymotrypsin, covalently modified alpha-chymotrypsin, and ribonuclease) in a plot of log k(in) versus reciprocal temperature (Arrhenius plot). These dependencies are characterized by the presence of both ascending and descending linear portions which have positive and negative values of the effective activation energy (Ea), respectively. A kinetic scheme has been suggested that fits best for a description of these zig-zag dependencies. A key element of this scheme is the temperature-dependent reversible conformational transition of enzyme from the 'low-temperature' native state to a 'high-temperature' denatured form; the latter form is significantly more stable against irreversible thermoinactivation than the native enzyme. A possible explanation for a difference in thermal stabilities is that low-temperature and high-temperature forms are inactivated according to different mechanisms. Existence of the suggested conformational transition was proved by the methods of fluorescence spectroscopy and differential scanning calorimetry. The values of delta H and delta S for this transition, determined from calorimetric experiments, are highly positive; this fact underlies a conclusion that this heat-induced transition is caused by an unfolding of the protein molecule. Surprisingly, in the unfolded high-temperature conformation, alpha-chymotrypsin has a pronounced proteolytic activity, although this activity is much smaller than that of the native enzyme.
authors.affiliation: Chemistry Department, M. V. Lomonosov Moscow State University, Russian Federation.
authors.lastname: Levitsky VYu
authors.forename: N S
authors.initials: NS
authors.lastname: Melik-Nubarov
authors.forename: V A
authors.initials: VA
authors.lastname: Siksnis
authors.lastname: Grinberg VYa
authors.forename: T V
authors.initials: TV
authors.lastname: Burova
authors.forename: A V
authors.initials: AV
authors.lastname: Levashov
authors.forename: V V
authors.initials: VV
authors.lastname: Mozhaev
mesh_terms: D002152:Calorimetry, Differential Scanning; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology
chemical_list: D004798:Enzymes; D013861:Thiocyanates; D012260:Ribonucleases; D002918:Chymotrypsin; C009941:potassium thiocyanate