Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

Ligand Binding and Linkage

Ligand Binding and Linkage

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...

Enzymes

Enzymes

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...

Induced-fit Model

Induced-fit Model

Most chemical reactions in cells require enzymes—biological catalysts that speed up the reaction without being consumed or permanently changed. They reduce the activation energy needed to convert the reactants into products. Enzymes are proteins, that usually work by binding to a substrate—a reactant molecule that they act upon.
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...

Allosteric Proteins-ATCase

Allosteric Proteins-ATCase

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...

Protein Complexes with Interchangeable Parts

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

Molecular dynamics simulations of beta-2-glycoprotein 1 (β2GPI) reveal that post-translational modifications facilitate novel structures.

Scientific reports·2026

Same author

Mycobacterium tuberculosis presenting as chest wall spindle cell neoplasm in an immunocompetent patient.

BMC infectious diseases·2026

Same author

Sexual dimorphism of early GPR183-dependent B cell responses in systemic lupus erythematosus.

iScience·2026

Same author

Hotspot Interactions between Two Fab Molecules in Molecular Dynamics Simulations Improve Predictive Models of Aggregation Kinetics.

Molecular pharmaceutics·2026

Same author

Systematic formulation optimisation to enhance buffer exchange recovery and storage stability for adeno-associated virus (AAV2) vectors.

International journal of pharmaceutics·2025

Same author

Stability Testing Reveals Photoselective Clipping of Heavy Chain C-Terminal Amino Acids That Leads to Fragmentation and Aggregation of an Antibody Fab Fragment.

Molecular pharmaceutics·2025

Search research articles

Related Experiment Video

Updated: Jul 5, 2025

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Published on: April 1, 2016

Two-substrate enzyme engineering using small libraries that combine the substrate preferences from two different

Arka Mukhopadhyay¹, Kersti Karu², Paul A Dalby³

¹Department of Biochemical Engineering, UCL, Bernard Katz Building, Gower Street, London, WC1E 6BT, UK.

Scientific Reports

|January 13, 2024

Summary

This summary is machine-generated.

Enzyme engineering advances biocatalysis by enabling new substrate combinations. Researchers successfully combined mutations to create novel enzyme variants with significantly improved activity for specific two-substrate reactions.

More Related Videos

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Published on: November 23, 2016

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Related Experiment Videos

Last Updated: Jul 5, 2025

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Published on: April 1, 2016

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Published on: November 23, 2016

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Area of Science:

Biocatalysis and enzyme engineering
Synthetic biology
Protein engineering

Background:

Enzyme industrialisation requires broader substrate scope and high catalytic activity.
Engineering enzymes for novel two-substrate reactions is complex due to interconnected active site roles.

Purpose of the Study:

To develop a method for combining beneficial mutations to reprogram enzyme active sites for new two-substrate reactions.
To enhance the catalytic activity of enzymes towards alternative substrates.

Main Methods:

Designed small mutant libraries incorporating natural and non-natural amino acids at key mutational sites.
Investigated enzyme variants for improved catalysis of the reaction between 3-formylbenzoic acid (3-FBA) and pyruvate.
Utilized computational docking to analyze active site structural changes and substrate-enzyme interactions.

Main Results:

Achieved up to a 630-fold increase in catalytic rate (kcat) for the 3-FBA and pyruvate reaction.
Identified specific mutations that enhance the proximity of 3-FBA to the enamine-Thiamine pyrophosphate (TPP) intermediate.
Demonstrated successful reprogramming of enzyme active sites for specific two-substrate conversions.

Conclusions:

Small libraries enable rapid, plug-and-play reprogramming of enzyme active sites.
This approach facilitates the creation of novel biocatalysts for diverse two-substrate reactions.
Advances enzyme engineering for industrial applications by expanding substrate acceptance and catalytic efficiency.