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Oligopeptide Competition Assay for Phosphorylation Site Determination
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How does the exosite of rhomboid protease affect substrate processing and inhibition?
Michael Shokhen1, Amnon Albeck1
1The Julius Spokojny Bioorganic Chemistry Laboratory, Department of Chemistry, Bar Ilan University, Ramat Gan, 5290002, Israel.
Protein Science : a Publication of the Protein Society
|September 9, 2017
Summary
Rhomboid proteases, a unique class of intramembrane enzymes, utilize substrate transmembrane domains for recognition. Molecular dynamics simulations reveal how GlpG rhomboid protease controls substrate hydrolysis and inhibitor binding.
Area of Science:
- Biochemistry
- Structural Biology
- Computational Biology
Background:
- Rhomboid proteases are intramembrane serine proteases with unique catalytic mechanisms distinct from soluble proteases.
- Understanding their function is crucial due to their ubiquitous presence and roles in various biological processes.
Purpose of the Study:
- To elucidate the catalytic mechanism of GlpG rhomboid protease using molecular dynamics (MD).
- To investigate substrate recognition, inhibitor binding, and specificity for transmembrane domain (TMD)-containing substrates.
Main Methods:
- Employed molecular dynamics (MD) simulations to analyze GlpG rhomboid protease.
- Focused on exosite interactions, active site (AS) inhibition, and substrate binding.
Main Results:
- The exosite's interaction with substrate TMDs dictates substrate hydrolysis kinetics.
- Non-competitive peptidyl aldehyde inhibitors bind to the GlpG active site, modulating activity.
- Rhomboid proteases show specificity for substrates containing TMDs, explaining limited hydrolysis of short peptides.
Conclusions:
- Substrate recognition via TMDs in the exosite is a fundamental feature of rhomboid catalysis.
- The enzyme transitions from a non-covalent pre-catalytic complex to a covalent tetrahedral complex (TC).
- These findings provide mechanistic insights into rhomboid protease function and substrate specificity.
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