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 (kJ of enzymes (a-chymotrypsin, covalently modified a-chymotrypsin, and ribonuclease) in a plot of log k,, 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 ( E J , 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 'lowtemperature' 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 AH and AS 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, a-chymotrypsin has a pronounced proteolytic activity, although this activity is much smaller than that of the native enzyme.It is well known that temperature dependencies of the rates of most reactions with enzyme participation, likewise usual chemical reactions, follow the Arrhenius equation [l]. Consequently, these temperature dependencies are strictly linear in a plot of log v (or log k) versus T', where v is the reaction rate, k is the corresponding rate constant, and T is absolute temperature. Deviations from the linear dependencies are not often encountered and are usually due to changes in the reaction mechanism or catalyst structure, or some other factors.Nonlinear temperature dependencies have sometimes also arisen in studies of the irreversible thermoinactivation of enzymes [2-41. Unlike reversible thermal denaturation, which usually is a single-step conformational transition between two states of a protein [5] recent reviews]. As a consequence, the apparent rate of irreversible thermoinactivation, v,,, (and the corresponding rate constant, kin) depends in a complicated manner on the rates of the processes which participate in thermal inactivation. These processes to different extents depend on temperature and this fact can explain appearance of nonlinear dependencies of v,, versus T-' [3, 41.In this study we observed temperature dependencies of the rates of irreversible thermal inactivation of a-chymotrypsin and ribonuclease of zig-zag type and have put forward a kinetic model that describes this unusual behavior. MATERIALS AND METHODS Materialsa-Chymotrypsin (Sigma,...

Correlation of high‐temperature stability of α‐chymotrypsin with ‘salting‐in’ properties of solution

“…However, in the preceding paper of this issue we have shown [28] that the long-term stability of a-chymotrypsin may depend on temperature in a rather different 'zig-zag' manner: the linear regions with positive values of the activation energy for thermoinactivation, E,, are separated by a region with negative apparent value of E,. Zig-zag dependencies were explained on the basis of the following scheme :…”

“…El is much less stable against irreversible thermal inactivation than E, [28]. The unfolding equilibrium between El and E, is strongly influenced by the addition of inorganic salts 1311 and other low-molecular-mass solutes [28]. Thus, on the basis of Eqn ( 3 ) , one can try to regulate the long-term stability of enzyme, for which the value of k,, is a quantitative measure, by changing the value of K by the addition of lowmolecular-mass compounds.…”

“…The rate constant of irreversible thermal inactivation of a-chymotrypsin, k,,, was determined from the linear plot of the kinetic curves, log AIA, versus t, where A and A, are the current and the initial (before inactivation) activities of the enzyme, t = time. When the kinetics of thermal inactivation had a biphasic character, k,, was determined from the slope of a flat portion of the second ('stable') phase [28]. In the experiments with additives, small concentrations of solutes (less than 0.1 M) which remained in the cuvette of the pH-stat after dilution of the enzyme solution, did not influence enzyme activity during an assay (15 min).…”

“…At high temperatures, protein transforms spontaneously from E, into E, as a result of the reversible conformational transition governed by the equilibrium constant K. The rate of inactivation of the enzyme is controlled by the apparent rate constant of thermoinactivation, k,,, which is given by the following combination of constants k,, k2, and K [28] :…”

“…At any temperature, k, is much higher than k2, i.e. El is much less stable against irreversible thermal inactivation than E, [28]. The unfolding equilibrium between El and E, is strongly influenced by the addition of inorganic salts 1311 and other low-molecular-mass solutes [28].…”