Temperature-dependent ΔC⁰_p generated by a shift in equilibrium between macrostates of an enzyme

Abstract

Substantial negative heat capacity changes (ΔC⁰_p′s) have frequently been observed to accompany the formation of protein–ligand complexes^1,2. Glutamate dehydrogenase³ and horse liver alcohol dehydrogenase⁴, however, have been reported to form binary complexes with coenzyme with negligible ΔH⁰′ and only small ΔC⁰_p′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 ΔC⁰_p′s of complex formation from the various mechanistic sources or even for distinguishing between them. However, if, as Eftink and Biltonen⁵ have suggested, a shift in equilibrium between macrostates contributes significantly to an observed ΔC⁰_p′s for a given reaction, it should be possible to characterize such a system by measuring the temperature dependence of the ΔC⁰_p′s. Despite this, few studies have determined ΔH⁰′ values at more than two temperatures. We have now measured the temperature dependence of the ΔH⁰′ (and, thereby, that of the ΔC⁰_p′s) of the formation of an enzyme-reduced coenzyme complex in an attempt to provide such a basis and have found that the entire ΔC⁰_p′s of complex formation is accounted for by a temperature-induced shift of an equilibrium between the different forms of the free enzyme.