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Synthesis of an Intein-mediated Artificial Protein Hydrogel
Published on: January 27, 2014
Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate
D J Vocadlo1, G J Davies, R Laine
1Protein Engineering Network of Centres of Excellence and the Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Nature
|August 24, 2001
Summary
Hen egg-white lysozyme (HEWL) catalysis involves a covalent intermediate, challenging the long-held Phillips mechanism. This study provides structural and mass spectrometry evidence for this intermediate in HEWL, refining our understanding of beta-glycosidase function.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Hen egg-white lysozyme (HEWL) was the first enzyme with a determined 3D structure.
- The proposed 'Phillips' mechanism for HEWL involves a long-lived oxocarbenium-ion intermediate.
- This mechanism is considered the paradigm for retaining beta-glycosidases.
Purpose of the Study:
- To investigate the catalytic mechanism of HEWL.
- To provide evidence for a covalent glycosyl-enzyme intermediate in HEWL's catalytic cycle.
- To formulate a general catalytic mechanism for retaining beta-glycosidases.
Main Methods:
- Electrospray ionization mass spectrometry (ESI-MS) to detect intermediates.
- X-ray diffraction to determine the 3D structure of the intermediate.
- Analysis of HEWL's catalytic cycle.
Main Results:
- Demonstrated a catalytically competent covalent glycosyl-enzyme intermediate in HEWL using ESI-MS.
- Determined the 3D structure of this covalent intermediate via X-ray diffraction.
- Observed this intermediate in three different cases.
Conclusions:
- The catalytic mechanism of HEWL involves a covalent glycosyl-enzyme intermediate.
- This finding supports a common mechanism for retaining beta-glycosidases.
- A general mechanism is proposed including substrate distortion, covalent intermediate formation, and C1 electrophilic migration.
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