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Enzyme Inhibition01:30

Enzyme Inhibition

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Inhibitors are molecules that reduce enzyme activity by binding to the enzyme. In a normally functioning cell, enzymes are regulated by a variety of inhibitors. Drugs and other toxins can also inhibit enzymes. Some inhibitors bind to the enzyme’s active site, while others inhibit enzymatic activity by binding to other sites on the protein structure.
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Enzymes02:34

Enzymes

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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...
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Induced-fit Model01:13

Induced-fit Model

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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...
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Ligand Binding and Linkage00:49

Ligand Binding and Linkage

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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
Allosteric Proteins-ATCase01:19

Allosteric 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...
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Allosteric Regulation01:08

Allosteric Regulation

59.0K
Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
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Related Experiment Video

Updated: Sep 8, 2025

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues
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Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

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Modifying inhibitor specificity for homologous enzymes by machine learning.

Dor S Gozlan1, Reut Meiri2, Gili Shapira1

  • 1Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

The FEBS Journal
|September 5, 2025
PubMed
Summary
This summary is machine-generated.

Machine learning streamlines selective protease inhibitor design. A novel N-TIMP2 variant showed enhanced selectivity for matrix metalloproteinases (MMPs), demonstrating reduced experimental effort and improved targeting of homologous enzymes.

Keywords:
deep mutational scanningmatrix metalloproteinasesneural networksprotein engineeringprotein–protein interactions

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Area of Science:

  • Biochemistry and Molecular Biology
  • Computational Biology
  • Drug Discovery

Background:

  • Selective enzyme inhibitors are crucial for targeted therapies and biological research.
  • Designing specific inhibitors, especially for homologous enzymes, faces challenges in experimental scale and specificity tuning.
  • Current machine learning (ML) approaches for protein design are limited by energy calculation accuracy and predicting multi-mutation effects.

Purpose of the Study:

  • To develop and validate a novel ML-based method for designing selective protease inhibitors.
  • To streamline the identification of inhibitors with tailored specificity profiles for homologous enzymes.
  • To apply the method to design selective inhibitors for matrix metalloproteinases (MMPs).

Main Methods:

  • Leveraging high-throughput screening (HTS) data with ML models to train predictive binding affinities.
  • Designing a novel N-TIMP2 variant targeting MMP-1, MMP-3, and MMP-9.
  • Experimental validation of the designed variant's binding affinity and selectivity.
  • Utilizing molecular modeling and energy minimization for structural insights.

Main Results:

  • Successfully designed a novel N-TIMP2 variant with a distinct specificity profile across MMP-1, MMP-3, and MMP-9.
  • Experimental validation confirmed a significant specificity shift and enhanced selectivity compared to wild-type N-TIMP2.
  • Structural analysis provided insights into the molecular basis of the variant's improved selectivity.

Conclusions:

  • The developed ML-based method effectively reduces experimental workload in inhibitor design.
  • The approach facilitates the rational design of highly selective inhibitors for homologous enzyme families.
  • This work advances the understanding of enzyme-inhibitor interactions and selective targeting.