Related Experiment Video
Updated: Sep 11, 2025
11:27
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
4.0K
Catalytic His-loop flexibility drives high activity in hyperthermophilic esterase EstE1 while preserving structural
Khang Nguyen1,2, ChangWoo Lee1,2
1Department of Biomedical Science, Daegu University, Gyeongsan, South Korea.
Microbiology Spectrum
|August 14, 2025
Summary
Hyperthermophilic esterase EstE1 achieves high activity through flexible catalytic His loops, unlike its mesophilic counterpart rPPE which relies on loop stabilization. Modulating loop flexibility offers insights for engineering stable, high-performance thermophilic enzymes.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Esterases are crucial enzymes catalyzing ester bond hydrolysis and formation, often utilizing a catalytic Ser-His-Asp triad.
- Hyperthermophilic esterase EstE1 displays high activity despite a rigid structure, contrasting with its mesophilic counterpart rPPE.
- The catalytic His loop's flexibility is investigated as a key modulator of enzyme activity in both EstE1 and rPPE.
Purpose of the Study:
- To investigate how the catalytic His loop influences enzyme activity in the hyperthermophilic esterase EstE1 and its mesophilic counterpart rPPE.
- To explore the structural basis for high catalytic activity in thermophilic enzymes through targeted mutagenesis.
- To provide insights into engineering thermophilic enzymes with enhanced performance and stability.
Main Methods:
- Site-directed mutagenesis was employed to introduce specific amino acid substitutions in EstE1 (Gly282 to Asn/Gln) and rPPE (Asp287 to Gly/Glu).
- Enzyme activity assays were conducted to quantify catalytic performance.
- Fluorescence spectroscopy and acrylamide quenching were used to assess protein structure, loop flexibility, and stability.
Main Results:
- Mutations in EstE1 (G282N, G282Q) that promoted or disrupted hydrogen bonding in the His loop reduced enzyme activity.
- EstE1's G282N mutant showed increased rigidity and time-dependent activity loss, while G282Q exhibited greater flexibility than wild-type.
- In rPPE, D287G enhanced activity via increased loop flexibility, and D287E improved stability and substrate affinity by strengthening hydrogen bonding.
Conclusions:
- Gly282 in EstE1 promotes His-loop flexibility essential for high-temperature activity, whereas rPPE utilizes His-loop hydrogen bonding for stability at the cost of activity.
- Modulating His-loop flexibility and stability through targeted mutations offers a strategy for enzyme engineering.
- Understanding these distinct mechanisms provides a framework for designing thermophilic enzymes with improved catalytic efficiency and structural integrity.
Related Concept Videos
01:13Introduction to Mechanisms of Enzyme Catalysis
8.8K
For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes...
8.8K
01:29Diversity of Archaea IV
105
Hyperthermophilic archaea are a group of extremophiles thriving at temperatures above 80°C, often in hydrothermal vents and volcanic soils where conditions surpass the boiling point of water. At such temperatures, proteins, membranes, and DNA in most organisms degrade, but hyperthermophiles have evolved remarkable adaptations to maintain stability and function.Unique Cellular FeaturesHyperthermophilic membranes are composed of a monolayer of biphytanyl tetraether lipids, which resist...
105
01:07Catalytically Perfect Enzymes
4.1K
The theory of catalytically perfect enzymes was first proposed by W.J. Albery and J. R. Knowles in 1976. These enzymes catalyze biochemical reactions at high-speed. Their catalytic efficiency values range from 108-109 M-1s-1. These enzymes are also called 'diffusion-controlled' as the only rate-limiting step in the catalysis is that of the substrate diffusion into the active site. Examples include triose phosphate isomerase, fumarase, and superoxide dismutase.
Most enzymes...
Most enzymes...
4.1K
01:19Allosteric Proteins-ATCase
5.9K
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
5.9K
00:49Ligand Binding and Linkage
4.9K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
4.9K
01:27Diversity of Archaea III
73
Crenarchaeota, a prominent phylum of Archaea, is remarkable for its ability to thrive in extreme environments characterized by high temperatures and acidity. These microorganisms inhabit sulfuric hot springs, volcanic systems, and submarine hydrothermal vents, where temperatures often exceed 100°C. The unique adaptations of Crenarchaeota not only allow survival under such extreme conditions but also provide insights into the mechanisms of life in primordial Earth-like...
73