Related Experiment Video
Updated: Dec 30, 2025
13:57
Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
Published on: February 18, 2014
30.1K
Reduced Enzyme Dynamics upon Multipoint Covalent Immobilization Leads to Stability-Activity Trade-off
James S Weltz1, Daniel F Kienle1, Daniel K Schwartz1
1Department of Chemical and Biological Engineering , University of Colorado Boulder , Boulder , Colorado 80309 , United States.
Journal of the American Chemical Society
|January 28, 2020
Summary
Multipoint covalent immobilization (MPCI) rigidifies enzymes, enhancing stability but reducing activity. Tuning enzyme attachment balances these effects for optimal biocatalyst design.
Area of Science:
- Biomaterials Science
- Enzyme Engineering
- Biocatalysis
Background:
- Multipoint covalent immobilization (MPCI) is crucial for enzyme applications in biocatalysis, biosensing, and defense.
- A clear mechanistic understanding of how MPCI affects enzyme stability is lacking.
- This knowledge gap limits the full potential of MPCI for enzyme stabilization.
Purpose of the Study:
- To elucidate the mechanistic basis of enzyme stabilization via MPCI.
- To investigate the impact of MPCI on enzyme structural dynamics and stability.
- To explore the relationship between enzyme stability, structural dynamics, and catalytic activity.
Main Methods:
- Enzyme (lipase) immobilization onto polymer brush surfaces using MPCI.
- Measurement of enzyme unfolding/refolding rates and structural fluctuations using single-molecule Förster Resonance Energy Transfer (SM-FRET) imaging.
- Assessment of enzyme activity and thermal stability by measuring activity at varying temperatures.
Main Results:
- MPCI significantly enhanced lipase stability by rigidifying the enzyme structure, evidenced by a 10-fold decrease in unfolding/refolding rates.
- Reduced intrinsic structural fluctuations in both folded and unfolded states were observed upon MPCI.
- A trade-off between enhanced thermal stability and decreased specific activity was identified; increased stability correlated with reduced catalytic efficiency.
Conclusions:
- Enzyme stabilization via MPCI results from structural rigidification and reduced dynamics.
- Decreased enzyme dynamics, while enhancing stability, negatively impacts catalytic activity by hindering essential motions.
- Tuning the extent of enzyme attachment offers a strategy to balance stability and activity in immobilized enzymes.
Related Concept Videos
01:58Cooperative Allosteric Transitions
8.6K
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
8.6K
01:58Cooperative Allosteric Transitions
2.6K
2.6K
01:13Introduction to Mechanisms of Enzyme Catalysis
10.3K
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...
10.3K
01:23Complexation Equilibria: Overview
1.2K
Complexation reactions take place when dative or coordinate covalent bonds form between metal ions and ligands. The compounds formed in these reactions are called coordination compounds. The number of bonds formed between the metal ion and the ligands is called its coordination number. Generally, most metal ions in an aqueous solution are solvated by water molecules and thus exist as aqua complexes.
The equilibrium constant of the complexation reaction is represented as the formation constant...
The equilibrium constant of the complexation reaction is represented as the formation constant...
1.2K
02:34Enzymes
92.4K
Inside living organisms, enzymes act as catalysts for many biochemical reactions involved in cellular metabolism. The role of enzymes is to reduce the activation energies of biochemical reactions by forming complexes with its substrates. The lowering of activation energies favor an increase in the rates of biochemical reactions.
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
92.4K
01:09Complexation Equilibria: Factors Influencing Stability of Complexes
724
In complexation reactions, metal cations are the electron pair acceptors, and the ligands are the electron pair donors. The stability of the metal complexes depends primarily on the complexing ability of the central metal ion and the nature of the ligands. Generally, the complexing ability of the metal ion depends on the size and charge of the ion. As the metal ion size increases, the stability of the metal complexes decreases, provided that the valency of the metal ion and the ligands remain...
724