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.
Abstract
Many bacterial chlorogenic acid esterases (ChlEs) exhibit atypical temperature behavior, featuring activities that barely change with temperature and activity maxima that fall below the thermal denaturation point. This work focuses on a ChlE fromLactobacillus helveticus (Lh–ChlE), which has a flat temperature dependence. First, it was determined that conformational changes during Lh–ChlE turnover are not rate-limiting and that the overall rate depends on the chemical step at all temperatures. Next, Lh–ChlE’s temperature dependence was investigated using a conformational equilibrium model that assumes the existence of a temperature-dependent equilibrium between an active and an inactive conformation and an activation heat capacity model that postulates a difference in heat capacity between the ground and transition states. Although the equilibrium model recapitulates the data well, it yields an unrealistically low inactivation temperature around 280 K. Circular dichroism spectroscopy suggests that Lh–ChlE does not undergo structural changes at that temperature but may undergo small structural transitions at moderately elevated temperature. The activation heat capacity model describes Lh–ChlE behavior well, yielding an activation heat capacity (ΔC p ‡) of approximately −1 kJ mol–1 K–1. Overall, the results suggest that the atypical temperature behavior of Lh–ChlE likely arises from a negative activation heat capacity. This work illustrates 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. Our results furthermore encourage additional analysis of the Lh–ChlE transition state structure to better understand the structural features that cause the enzyme’s nonzero activation heat capacity.
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.
Ignacio Aravena-Valenzuela, P. Maturana, Leslie Hernández-Cabello et al.· bioRxiv· 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
Microbial esterases are versatile and stable enzymes with a wide range of biotechnological applications. However, few esterases have been characterized from archaea, an important source of extremophilic enzymes. In this study, we report the biochemical characterization and crystal structure of Ta0887, a novel esterase from the thermoacidophilic archaeon Thermoplasma acidophilum. The protein was successfully cloned, expressed, and purified in Escherichia coli. Light scattering assays revealed that Ta0887 is a monomer in solution. Activity assays using p‐nitrophenyl (p‐NP) esters confirmed its esterase activity, showing a substrate preference for p‐NP hexanoate (C6). Furthermore, the substitution of Ser95 with alanine completely abolished enzymatic activity, thereby confirming its essential role as the nucleophilic residue of the catalytic triad. The enzyme exhibited optimal activity at 65 °C and pH 8.0. Notably, Ta0887 displayed high thermal stability, retaining 66% residual activity after incubation at 80 °C for 2 h, consistent with its thermal denaturation midpoint temperature of 80.6 °C. The crystal structure of Ta0887, resolved at 1.93 Å, revealed an α/β‐hydrolase core domain consisting of an eight‐strand β‐sheet, surrounded by seven α‐helices, and a cap domain comprising four α‐helices. Ta0887 features a large substrate‐binding pocket at the interface between the two domains that contains the conserved residues Ser95, Asp187, and His215 of the catalytic triad. Further analysis indicates that an efficiently packed hydrophobic core is a key feature for the observed thermostability. The findings from this study provide a basis for the future engineering of Ta0887 with the aim of enhancing its potential for industrial and biotechnological applications.
Alejandro Delgado-Rey, M. L. Llamas-García, Gabriela M Montero-Morán et al.· FEBS Open Bio· 0 citations
The stability and inactivation of (S)-1-phenylethanol dehydrogenase (S-PEDH) were investigated over a broad pH range under storage and reactor conditions. The enzyme displayed distinct pH optima depending on the parameter considered, with maximal activity at acidic pH (3.5-5.5), highest operational stability at alkaline pH (8.3-9.0), and maximal thermal stability at near-neutral pH. Ligand-dependent effects were observed, with NADH increasing the melting temperature, while isopropanol and acetophenone caused destabilization and reduced catalytic performance. Thermal inactivation was experimentally shown to follow a biphasic pattern. Combined analysis of enzymatic activity and hydrodynamic radius supports a mechanism in which conformational destabilization precedes aggregate growth; suggesting that aggregation a secondary consequence of the initial inactivation event rather than its primary cause. The results of our study highlight the importance of balancing pH, temperature, and solvent conditions to optimize enzyme performance in biocatalytic applications.
Mateusz Tataruch, V. Illeová, Anna Kluza et al.· International Journal of Bio...· 0 citations
The results show that enzyme unfolding is irreversible and kinetically controlled and is best described by a three-state Lumry-Eyring model involving a reversible intermediate followed by an irreversible transition to an aggregated stated, likely contributing to enzyme robustness under the dynamic conditions encountered during host-pathogen interactions.
Daniela S. Barreiro, S. R. Pauleta· Archives of Biochemistry and...· 0 citations
