This work characterized the glucokinase activity of three homologous bifunctional ADP-dependent PFK/GK enzymes and presented a two-pathway model incorporating a conformational equilibrium in which free enzyme and enzyme-substrate complex populate two catalytically competent conformations.
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 , 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.
It is suggested that contrasting thermodynamic models for atypical temperature dependence in enzymes can give rise to similar looking fits, even though they have different underlying physical meaning.
Nathaniel B. Carl, Yianni Tsigaris, Dan Ji et al.· Biochemistry· 0 citations
It is suggested that cold adaptation in pBGL may involve not only enhanced flexibility but also network-mediated organization of conformational dynamics, providing a structural framework for understanding the activity–stability balance of cold-active enzymes.
Xian He, Mengting Liu, Xintong Li et al.· Frontiers in Microbiology· 1 citation
A stability study of small cold shock proteins from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima finds that cold unfolding appears as a new but unsuspected factor limiting life at low temperatures.
Lorenzo Grossi, Caroline Struvay, G. Feller· Biochimie· 0 citations
Substrate specificity in lactate dehydrogenases (LDHs) controls metabolic flux and remains a central challenge in enzyme engineering, particularly when closely related substrates differ by only a single carboxylate group. Although Q102 (equivalent to Gln86 in Geobacillus stearothermophilus L-LDH) is recognized as a canonical substrate-specificity determinant, how this electrostatic switch cooperates with steric effects, conformational dynamics, and scaffold context to govern substrate recognition remains unclear. Here, we combined steady-state kinetics with molecular dynamics simulations to examine specificity switching from pyruvate to oxaloacetate in G. stearothermophilus L-LDH (GsLDH). The Q86R substitution enhanced oxaloacetate catalytic efficiency 34-fold (kcat/Km = 340 s-1 mM-1) through favorable electrostatic stabilization and increased substrate retention, but substantially reduced pyruvate activity. In contrast, I227V acted primarily through steric accommodation, broadening substrate acceptance by retaining substantial pyruvate activity (377 s-1 mM-1) while markedly increasing oxaloacetate activity (322 s-1 mM-1). The combined Q86R/I227V variant displayed positive epistasis and achieved the highest oxaloacetate efficiency among engineered LDH variants (738 s-1 mM-1), approaching that of native G. stearothermophilus malate dehydrogenase (GsMDH) (771 s-1 mM-1), indicating complementary contributions of electrostatic stabilization and steric optimization. Loop-state and productive-geometry analyses further showed that loop closure alone is permissive but insufficient for catalysis; productive turnover requires coupling between loop conformation, substrate retention, and hydride/proton-transfer geometry. Principal component analysis further revealed that the engineered mutations redistribute the conformational ensembles toward substrate-compatible productive states. Reciprocal mutations GsMDH failed to restore efficient pyruvate catalysis, demonstrating that specificity switching is asymmetric and constrained by scaffold context. Collectively, these findings support an electrostatic-steric ensemble-selection model for LDH/MDH specificity and provide a mechanistic framework for engineering substrate specificity in Rossmann-fold dehydrogenases.
Hanfeng Cai, S. Shulami, T. Hrenar et al.· International Journal of Bio...· 0 citations
The computed C p profile of the unmodified tRNA displayed a well-defined peak consistent with experimentally reported C p thermograms for various tRNAs, and the effects of different monovalent ions, distinct interatomic potentials, and varying melting temperature values on the key thermophysical properties of the tRNA isodecoder were probed.
Lev Levintov, Esteban A. Orellana, Harish Vashisth· Journal of Physical Chemistr...· 0 citations
Protein dynamism and evolvability are key parameters linking enzyme flexibility to adaptive potential, yet how these concepts apply to nucleic acid enzymes remains largely unexplored. Here, we propose that threose nucleic acid (TNA), a genetic polymer that is more conformationally restricted than DNA, may be evolutionarily constrained by its preorganized backbone. RNA-cleavage profiles comparing the well-known 10-23 DNA enzyme (DNAzyme) with two in vitro selected TNA enzymes (threozymes) reveal striking differences in their temperature dependence. While the DNAzyme catalyzed reaction is optimal at 37 °C and weakly active at 50 °C, threozymes display the opposite trend, suggesting that TNA catalysis must overcome a higher free-energy barrier than DNA catalysis. Consistent with this view, the reaction becomes less temperature dependent for threozymes that operate with more flexible active sites. Together, these findings highlight the importance of backbone structure as a critical parameter for evolvability with implications for RNA world models and biomedical applications.
Erica Kim Man Lee, John C. Chaput· Nature Communications· 0 citations
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