Two activation heat capacity regimes underlie temperature-dependent catalysis in homologous archaeal ADP-dependent kinases
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 , reflecting a transition-state ensemble more conformationally restricted than the ground state. Recently, 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 Methanococcoides burtonii, MmPFK/GK from the mesophilic Methanococcus maripaludis, and ancM, the inferred ancestor of the Methanococcales order, which displays enhanced thermostability. MmPFK/GK and ancM display two regimes, with abrupt changes in kcat vs temperature: zero to moderately negative values at low temperatures, shifting sharply at elevated temperatures to highly negative values (−44 kJ mol−1 K−1 and −36 kJ mol−1 K−1, 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 and a single regime throughout all temperatures (−2.6 kJ mol−1 K−1). Domain-closure dynamics explain thermal adaptation and moderate-temperature values; whereas the basis of the extreme high-temperature 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.