Two activation heat capacity regimes underlie temperature-dependent catalysis in homologous archaeal ADP-dependent kinases

Aravena-Valenzuela, I.; Maturana, P.; Hernandez-Cabello, L.; Gonzalez-Ordenes, F.; Castro-Fernandez, V.; Vallejos-Baccelliere, G.; Guixe, V.

doi:10.64898/2026.08.05.742859

bioRxiv · 10.64898/2026.08.05.742859

Two activation heat capacity regimes underlie temperature-dependent catalysis in homologous archaeal ADP-dependent kinases

Aravena-Valenzuela, I.·Maturana, P.·Hernandez-Cabello, L.·Gonzalez-Ordenes, F.·Castro-Fernandez, V.·Vallejos-Baccelliere, G.·Guixe, V.

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Abstract

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 [Formula], reflecting a transition-state ensemble more conformationally restricted than the ground state. Recently, [Formula] 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 [Formula] 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 [Formula] and a single [Formula] regime throughout all temperatures (-2.6 kJ mol-1 K-1). Domain-closure dynamics explain thermal adaptation and moderate-temperature [Formula] values; whereas the basis of the extreme high-temperature [Formula] 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.

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Aravena-Valenzuela, I., Maturana, P., Hernandez-Cabello, L., Gonzalez-Ordenes, F., Castro-Fernandez, V., Vallejos-Baccelliere, G., Guixe, V.. 2026-08-06. Two activation heat capacity regimes underlie temperature-dependent catalysis in homologous archaeal ADP-dependent kinases. https://doi.org/10.64898/2026.08.05.742859

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