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

Engineering enzyme specificity by "substrate-assisted catalysis".

P Carter, J A Wells

    Science (New York, N.Y.)
    |July 24, 1987
    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

    Best-Practice Biomarker Testing of Oesophago-Gastric Cancer in the UK: Expert Consensus Recommendations Developed Using a Modified Delphi.

    Clinical oncology (Royal College of Radiologists (Great Britain))·2024
    Same author

    Peri-operative management and analgesic strategy for a patient undergoing quadruple limb amputation.

    Anaesthesia reports·2024
    Same author

    An assessment of introducers used for airway management.

    Anaesthesia·2021
    Same author

    A demonstration of using formal consensus methods within guideline development; a case study.

    BMC medical research methodology·2021
    Same author

    Non-Invasive MRI of Blood-Cerebrospinal Fluid Barrier Function.

    Nature communications·2020
    Same author

    Management of mesh complications following surgery for stress urinary incontinence or pelvic organ prolapse: a systematic review.

    BJOG : an international journal of obstetrics and gynaecology·2019
    Same journal

    A native sulfur deposit in Gale crater, Mars.

    Science (New York, N.Y.)·2026
    Same journal

    Coordinated demise of harmful algal blooms.

    Science (New York, N.Y.)·2026
    Same journal

    Genetic effects put into context.

    Science (New York, N.Y.)·2026
    Same journal

    Bacteria share proteins to survive antibiotics.

    Science (New York, N.Y.)·2026
    Same journal

    Impacts shaped Earth's first continents.

    Science (New York, N.Y.)·2026
    Same journal

    Erratum for the Report "Covalently bonded single-molecule junctions with stable and reversible photoswitched conductivity" by C. Jia <i>et al</i>.

    Science (New York, N.Y.)·2026
    See all related articles

    Researchers engineered enzyme specificity by removing a catalytic group and restoring activity with a modified substrate. This "substrate-assisted catalysis" offers a new method for creating highly specific enzymes.

    Area of Science:

    • Biochemistry
    • Enzyme Engineering
    • Molecular Biology

    Background:

    • Enzyme specificity is crucial for biological functions.
    • Engineering enzyme specificity is a significant challenge in biotechnology.
    • Site-directed mutagenesis is a common tool for enzyme modification.

    Purpose of the Study:

    • To develop a novel approach for engineering enzyme specificity.
    • To investigate the concept of "substrate-assisted catalysis".

    Main Methods:

    • Site-directed mutagenesis was used to replace catalytic Histidine (His) with Alanine (Ala) in Bacillus amyloliquefaciens subtilisin (His64Ala mutant).
    • Enzyme activity was assayed using various substrates, including N-succinyl-L-Phe-L-Ala-L-Ala-L-Phe-p-nitroanilide (sFAAF-pNA), N-succinyl-L-Phe-L-His-L-Ala-L-Phe-p-nitroanilide (sFAHF-pNA), and N-succinyl-L-Phe-L-Gln-L-Ala-L-Phe-p-nitroanilide (sFAQF-pNA).

    Related Experiment Videos

  • Substrate-dependent pH profiles and hydrolysis of large polypeptides were analyzed.

    • Main Results:

    • The His64Ala mutant showed a million-fold decrease in catalytic efficiency with sFAAF-pNA compared to wild-type subtilisin.
    • The His64Ala mutant hydrolyzed a His P2 substrate (sFAHF-pNA) up to 400 times faster than Ala P2 or Gln P2 substrates.
    • Wild-type subtilisin hydrolyzed these three substrates with similar catalytic efficiencies.
    • The His64Ala mutant demonstrated partial recovery of function by utilizing the substrate's histidine side chain.

    Conclusions:

    • "Substrate-assisted catalysis" is a viable strategy for engineering narrow and useful enzyme specificities.
    • This approach provides a new basis for enzyme design and modification.
    • The findings suggest a potential evolutionary intermediate for the catalytic triad of serine proteases.