Temperature-dependent ΔC0p generated by a shift in equilibrium between macrostates of an enzyme

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Abstract
Substantial negative heat capacity changes (delta C0p' s) have frequently been observed to accompany the formation of protein-ligand complexes. Glutamate dehydrogenase and horse liver alcohol dehydrogenase, however, have been reported to form binary complexes with coenzyme with negligible delta H0p' and only small delta C0p' s. Although many intriguing mechanisms have been proposed to account for the observed phenomena, there is little direct experimental evidence available which might provide a basis for evaluating the contributions of delta C0p' s of complex formation from the various mechanistic sources or even for distinguishing between them. However, if, as Eftink and Biltonen have suggested, a shift in equilibrium between macrostates contributes significantly to an observed delta C0p' s for a given reaction, it should be possible to characterize such a system by measuring the temperature dependence of the delta C0p'. Despite this, few studies have determined delta H0' values at more than two temperatures. We have now measured the temperature dependence of the delta H0' (and, thereby, that of the delta C0p') of the formation of an enzyme-reduced coenzyme complex in an attempt to provide such a basis and have found that the entire delta C0p' of complex formation is accounted for by a temperature-induced shift of an equilibrium between the different forms of the free enzyme.