Abstract
<jats:p> Enzyme activity increases with temperature up to a maximum, beyond which it declines, a behaviour traditionally attributed to thermal denaturation. However, some enzymes show activity decline well below the melting temperature. Macromolecular rate theory (MMRT) explains this phenomenon by introducing a negative activation heat capacity (ΔC <jats:sup>‡</jats:sup> <jats:sub>P</jats:sub> ), reflecting a transition-state ensemble more conformationally restricted than the ground state. Recently, ΔC <jats:sup>‡</jats:sup> <jats:sub>P</jats:sub> has been shown to be temperature-dependent and proposed as a general catalytic feature, though its variation within and across homologous families from distinct thermal niches remains unexplored. We characterized the glucokinase activity of three homologous bifunctional ADP-dependent PFK/GK enzymes: MbPFK/GK from the psychrotolerant <jats:italic>Methanococcoides burtonii</jats:italic> , MmPFK/GK from the mesophilic <jats:italic>Methanococcus maripaludis</jats:italic> , and ancM, the inferred ancestor of the <jats:italic>Methanococcales</jats:italic> order, which displays enhanced thermostability. MmPFK/GK and ancM display two ΔC <jats:sup>‡</jats:sup> <jats:sub>P</jats:sub> regimes, with abrupt changes in k <jats:sub>cat</jats:sub> vs temperature: zero to moderately negative values at low temperatures, shifting sharply at elevated temperatures to highly negative values (−44 kJ mol <jats:sup>−1</jats:sup> K <jats:sup>−1</jats:sup> and −36 kJ mol <jats:sup>−1</jats:sup> K <jats:sup>−1</jats:sup> , respectively), exceeding previous reports. Circular dichroism spectroscopy confirms that these extreme values reflect pre-melting conformational changes rather than denaturation. Despite being psychrotolerant, MbPFK/GK displayed the highest thermal stability (T <jats:sub>m</jats:sub> <jats:sup>app</jats:sup> = 87 °C) and a single ΔC <jats:sup>‡</jats:sup> <jats:sub>P</jats:sub> regime throughout all temperatures (−2.6 kJ mol <jats:sup>−1</jats:sup> K <jats:sup>−1</jats:sup> ). Domain-closure dynamics explain thermal adaptation and moderate-temperature ΔC <jats:sup>‡</jats:sup> <jats:sub>P</jats:sub> values; whereas the basis of the extreme high-temperature ΔC <jats:sup>‡</jats:sup> <jats:sub>P</jats:sub> values remain unknown. To account for these two regimes, we present a two-pathway model incorporating a conformational equilibrium in which free enzyme and enzyme-substrate complex populate two catalytically competent conformations. </jats:p>