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

<jats:title>Abstract</jats:title> <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 <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="742859v1_inline1.gif"/> </jats:inline-formula> , reflecting a transition-state ensemble more conformationally restricted than the ground state. Recently, <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="742859v1_inline2.gif"/> </jats:inline-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 <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 <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="742859v1_inline3.gif"/> </jats:inline-formula> regimes, with abrupt changes in <jats:italic>k</jats:italic> <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 <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="742859v1_inline4.gif"/> </jats:inline-formula> and a single <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="742859v1_inline5.gif"/> </jats:inline-formula> 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 <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="742859v1_inline6.gif"/> </jats:inline-formula> values; whereas the basis of the extreme high-temperature <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="742859v1_inline7.gif"/> </jats:inline-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. </jats:p>