Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

A large increase in enzyme-substrate affinity by protein engineering.

A J Wilkinson, A R Fersht, D M Blow

    Nature
    |January 12, 1984
    PubMed
    Summary
    This summary is machine-generated.

    Related Concept Videos

    You might also read

    Related Articles

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

    Sort by
    Same author

    Indexing electron backscatter diffraction patterns with a refined template matching approach.

    Ultramicroscopy·2019
    Same author

    Validation of an algorithm to reveal the U wave in atrial fibrillation.

    Scientific reports·2018
    Same author

    Quantitative imaging of anti-phase domains by polarity sensitive orientation mapping using electron backscatter diffraction.

    Scientific reports·2017
    Same author

    The dissolvable bead: A novel in vitro biofilm model for evaluating antimicrobial resistance.

    Journal of microbiological methods·2017
    Same author

    Diffraction effects and inelastic electron transport in angle-resolved microscopic imaging applications.

    Journal of microscopy·2017
    Same author

    Assessing the precision of strain measurements using electron backscatter diffraction--part 2: experimental demonstration.

    Ultramicroscopy·2013
    Same journal

    Daily briefing: How cooperation built the world.

    Nature·2026
    Same journal

    Deep-sea oddities and boatloads of other new species - June's best science images.

    Nature·2026
    Same journal

    From cloning to gene-editing: the enduring legacy of Dolly the sheep.

    Nature·2026
    Same journal

    Time to give hydration breaks the red card? What science says about keeping cool.

    Nature·2026
    Same journal

    Universities are relying on AI-detection software to catch cheating. How well do the programs work?

    Nature·2026
    Same journal

    Daily briefing: 'Cyborg' cockroaches breathe underwater with printed suit.

    Nature·2026
    See all related articles

    Protein engineering via site-directed mutagenesis significantly enhanced tyrosyl-tRNA synthetase activity. A Proline mutation dramatically improved enzyme-substrate affinity for ATP, demonstrating the power of in vitro mutagenesis.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzyme Engineering

    Background:

    • Tyrosyl-tRNA synthetase (TyrRS) is crucial for protein synthesis.
    • A specific threonine residue (Thr 51) in TyrRS was hypothesized to hinder substrate binding.
    • Understanding enzyme-substrate interactions is key to protein engineering.

    Purpose of the Study:

    • To engineer tyrosyl-tRNA synthetase for improved ATP binding affinity.
    • To investigate the role of Thr 51 in enzyme-substrate complex stability.
    • To demonstrate the utility of in vitro mutagenesis for enzyme optimization.

    Main Methods:

    • Oligodeoxynucleotide-directed mutagenesis was used to create Thr 51 Ala and Thr 51 Pro mutants.
    • Enzyme kinetics were measured to assess changes in activity (kcat/KM) and substrate affinity (KM).

    Related Experiment Videos

  • Crystallographic data informed the structural hypothesis regarding Thr 51.

    • Main Results:

    • Both Thr 51 Ala and Thr 51 Pro mutants exhibited increased catalytic activity (kcat/KM) for ATP.
    • The Thr 51 Pro mutant showed a significant 25-fold increase in overall activity.
    • This enhancement was primarily attributed to a substantially lowered Michaelis constant (KM) for ATP in the Pro 51 mutant.

    Conclusions:

    • Mutating Thr 51 to Proline in tyrosyl-tRNA synthetase dramatically improves ATP binding affinity.
    • This study validates in vitro mutagenesis as a powerful strategy for enhancing enzyme function.
    • Targeting specific residues can effectively overcome kinetic barriers in enzyme-substrate interactions.