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

Trypsin specificity increased through substrate-assisted catalysis

D R Corey1, W S Willett, G S Coombs

  • 1Howard Hughes Medical Institute, Department of Pharmacology, and Molecular Biophysics, University of Texas Southwestern Medical Center at Dallas 75235, USA.

Biochemistry
|September 12, 1995
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

Structure of an affinity-matured inhibitory recombinant fab against urokinase plasminogen activator reveals basis of potency and specificity.

Biochimica et biophysica acta. Proteins and proteomics·2020
Same author

Modulation of Wnt/β-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer.

Oncogene·2011
Same author

Two approaches to discovering and developing new drugs for Chagas disease.

Memorias do Instituto Oswaldo Cruz·2009
Same author

Substrate specificity profiling and identification of a new class of inhibitor for the major protease of the SARS coronavirus.

Biochemistry·2007
Same author

Telomerase-dependent reactivation of DNA synthesis in macrophages implies alteration of telomeres.

Cell biology international·2002
Same author

Engineering orthogonal ligand-receptor pairs from "near drugs".

Journal of the American Chemical Society·2001
Same journal

Aromatic Cage-Directed Azide-Methyllysine Photochemistry for Profiling Nonhistone Interacting Partners of the MeCP2 Methyl-CpG-Binding Domain.

Biochemistry·2026
Same journal

Differential Hydroxypyruvate Processing by <i>E. coli</i> and <i>P. aeruginosa</i> DXP Synthases Reveals Preferential Xylulose 5-Phosphate Formation by the <i>P. aeruginosa</i> Enzyme.

Biochemistry·2026
Same journal

Structural and Functional Characterization of Heterologous Nitrogenase Complexes.

Biochemistry·2026
Same journal

Discovery of Bacterial Unspecific Peroxygenases.

Biochemistry·2026
Same journal

Lactate Biology: Subcellular Routing and Chemical Form Define Function.

Biochemistry·2026
Same journal

Nature's Anaerobic Toolkit: Glycyl Radical Enzymes and Their Expanding Functional and Mechanistic Diversity.

Biochemistry·2026
See all related articles

Replacing histidine 57 with alanine in trypsin (H57A) enabled substrate-assisted catalysis, creating novel protease specificities. This modified trypsin selectively cleaves folded proteins, offering new tools for protein hydrolysis.

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Chemistry

Background:

  • The catalytic triad, including Histidine 57 (H57), is crucial for serine protease activity.
  • Investigating substrate-assisted catalysis offers a route to engineer novel enzyme specificities.

Purpose of the Study:

  • To determine if trypsin variant H57A, with H57 replaced by alanine, can perform substrate-assisted catalysis.
  • To characterize the substrate specificity and cleavage efficiency of trypsin H57A.
  • To explore the potential applications of trypsin H57A in selective protein hydrolysis.

Main Methods:

  • Site-directed mutagenesis was used to create the trypsin H57A variant.
  • Kinetic parameters (kcat/Km) were measured for various peptide substrates.
  • Cleavage of folded ornithine decarboxylase and a trypsinogen propeptide was assessed.

Related Experiment Videos

Main Results:

  • Trypsin H57A exhibited enhanced catalytic efficiency (kcat/Km) with specific peptide substrates containing histidine.
  • New protease specificities were generated, favoring sequences with histidine at P2 or P1'.
  • Trypsin H57A demonstrated selective cleavage of folded ornithine decarboxylase and efficiently processed a trypsinogen variant propeptide.

Conclusions:

  • Histidine 57 is not essential for substrate-assisted catalysis in trypsin; alanine substitution can facilitate this mechanism.
  • Trypsin H57A exhibits unique substrate specificities and can cleave folded proteins, expanding its utility.
  • The findings suggest that substrate-assisted catalysis can be extended to other serine proteases, enabling tailored protein hydrolysis strategies.