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Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase
Published on: December 19, 2011
Capturing acyl-enzyme intermediates with genetically encoded 2,3-diaminopropionic acid for hydrolase substrate
1Department of Oncology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Centre for Advanced Interdisciplinary Science and Biomedicine of IHM, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.
This study introduces a novel method using 2,3-diaminopropionic acid (DAP)-containing hydrolase mutants to trap and identify enzyme substrates. This technique enables the discovery of serine or cysteine hydrolase substrates in various cell types.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Cysteine and serine hydrolases play crucial roles in biological processes.
- Identifying their substrates is essential for understanding enzyme function.
- Current methods for substrate identification can be challenging, especially in complex biological systems.
Purpose of the Study:
- To provide a detailed protocol for substrate trapping and identification using genetically encoded 2,3-diaminopropionic acid (DAP)-containing hydrolase mutants.
- To demonstrate the application of this method for identifying substrates of the human herpesvirus 1 UL36USP peptidase domain.
- To enable substrate identification in both cell lysates and live mammalian cells.
Main Methods:
- Development of hydrolase mutants with catalytic cysteine or serine replaced by DAP.
- Capture of transient acyl-enzyme intermediates as stable amide analogs.
- Downstream identification of trapped substrate fragments using mass spectrometry and immunoblotting.
- Detailed step-by-step procedures for substrate trapping in cell lysate and live mammalian cells (adherent and suspension).
Main Results:
- Successful demonstration of DAP-mediated substrate trapping and identification.
- Identification of substrates for the human herpesvirus 1 UL36USP peptidase domain.
- Protocol adaptable for various cell types including bacteria, yeasts, and mammalian cells.
Conclusions:
- DAP-mediated substrate trapping is a versatile and effective method for identifying serine and cysteine hydrolase substrates.
- This protocol facilitates substrate discovery in complex mixtures and live cells.
- The method offers a practical approach for advancing research in enzymology and virology.
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