Biology subjects

Gonzalez-Ordenes, F.

Publications and source records attributed to Gonzalez-Ordenes, F..

2 recordsLinked to original sources

bioRxiv2026

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 [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.

biochemistry↗

bioRxiv2024

Glycogen phosphorylase from the methanogenic archaeon Methanococcus maripaludis: Unique regulatory properties of a pyridoxal 5'-phosphate independent phosphorylase

Glycogen phosphorylase (GP) is a critical enzyme in glycogen metabolism. Even though methanogens from the archaeal orders Methanosarcinales and Methanococcales are unable to grow on sugars, they store glycogen, which is metabolized through the glycogenolysis and glycolytic pathways when the carbon source for methanogenesis is depleted. Under these metabolic conditions, the activity of the GP enzyme is essential. To be active, all phosphorylases characterized to date require the cofactor pyridoxal 5-phosphate (PLP). This cofactor is covalently bound via Schiff base to a strictly conserved lysine residue at the active site. Extensive GP sequence analysis of organisms from different domains of life shows strict conservation of active site residues despite significant differences in sequence length. Interestingly, in GP sequences of organisms from the order Methanococcales of archaea, a threonine residue replaces the conserved lysine involved in PLP binding. The purification and characterization of recombinant GP from Methanococcus maripaludis show that the enzyme exhibits glycogen phosphorylase activity and high specificity for glycogen as a substrate. Analysis of the PLP content performed by several methods, such as absorbance, fluorescence, cyanohydrin adduct formation, and mass spectrometry, confirmed the absence of PLP. The results demonstrate that an archaeal GP from the order Methanococcales performs catalysis without the PLP cofactor, deviating from the well-established phosphorylase catalytic mechanism and revealing new scenarios for the glucosyltransferase reaction. Moreover, analysis of enzyme regulation shows that the activity is affected by various molecules, including nucleotides, intermediates of carbon metabolism, and phosphate species. Most of these molecules have not previously been identified as regulators of glycogen phosphorylases in prokaryotes. These results suggest that other GPs from Methanococcales can undergo complex regulation.

biochemistry↗