Testing Macromolecular Rate Theory

Enzymatic rate increase with respect to temperature has widely been described by transition-state theory. The experimentally observed rate decline above an optimum temperature (Tₒₚₜ) for enzymes has previously been attributed to thermal denaturation, despite the known discrepancies with this rationalisation. Recently, a new model has been proposed to describe the temperature dependence of enzymatic rates: macromolecular rate theory (MMRT). This new theory incorporates heat capacity into the rate equation to provide a more robust thermodynamic description of rates, and account for the distinct curvature seen in biological temperature-rate profiles. The current study explores the effect of enzyme vibrational modes on heat capacity, and how alterations to the distribution of these modes, by heavy isotope substitution, affect the change in heat capacity along the reaction coordinate. The results presented show clear evidence for the role of vibrational mode frequency distributions in governing the curvature of temperature-rate profiles, and present a hypothesis for how this shift differs for the enzyme-substrate complex and the enzyme-transition state complex. The study also addresses the ability of MMRT to accurately model enzymatic rates over a wide temperature range for two different enzymes. As a result of this study, a new MMRT equation was generated to include a temperature dependence term for the heat capacity, a factor previously considered negligible. The data generated has shown that heat capacity is temperature dependent and this is a significant factor in accurately describing enzymatic rates with respect to temperature. In particular, for complex enzymatic reactions whose enthalpy distribution is significantly narrowed over the reaction co-ordinate. These findings have helped to develop MMRT to provide more accurate descriptions of a broader range of data. Additionally, the conclusions regarding vibrational mode distribution provide insight into the physical basis for heat capacity changes over the course of the enzymatic reaction. Investigation of the hypotheses generated from this research will offer further insight into the mechanistic contributions of vibrational modes and heat capacity in enzyme catalysis