Uncovering the Relationship between the Change in Heat Capacity for Enzyme Catalysis and Vibrational Frequency through Isotope Effect Studies

Understanding how enzyme catalysis varies with temperature is key to understanding catalysis itself and, ultimately, how to tune temperature optima. Temperature dependence studies inform on the change in heat capacity during the reaction, ΔC P ‡, and we have recently demonstrated that this can expose links between the protein free energy landscape and enzyme turnover. By quantifying ΔC P ‡, we capture information on the changes to the distribution of vibrational frequencies during enzyme turnover. The primary experimental tool to probe the role of vibrational modes in a chemical/biological process is isotope effect measurements, since isotopic substitution primarily affects the frequency of vibrational modes at/local to the position of isotopic substitution. We have monitored the temperature dependence of a range of isotope effects on the turnover of a hyper-thermophilic glucose dehydrogenase. We find a progressive effect on the magnitude of ΔC P ‡ with increasing isotopic substitution of d-glucose. Our experimental findings, combined with molecular dynamics simulations and quantum mechanical calculations, demonstrate that ΔC P ‡ is sensitive to isotopic substitution. The magnitude of the change in ΔC P ‡ due to substrate isotopic substitution indicates that small changes in substrate vibrational modes are “translated” into relatively large changes in the (distribution and/or magnitude of) enzyme vibrational modes along the reaction. Therefore, the data suggest that relatively small substrate isotopic changes are causing a significant change in the temperature dependence of enzymatic rates.