Labelling of the catalytic site of lysozyme |
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| Authors: | G L Rossi E Holler S Kumar J A Rupley G P Hess |
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| Affiliation: | 1. State Key Laboratory of Modern Optical Instrumentations, Centre for Optical and Electromagnetic Research, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310058, China;2. Department of Chemistry, Institute of Molecular Functional Materials, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China;3. Guangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China;1. Synthetic Organic and Medicinal Chemistry Lab, Centre for Molecular and Nanomedical Sciences, International Research Centre, Sathyabama Institute of Science and Technology (Deemed to be University), Chennai 600119, Tamil Nadu, India;2. School of Bio and Chemical Engineering, Department of Biotechnology, Sathyabama Institute of Science and Technology (Deemed to be University), Chennai 600119, Tamil Nadu, India |
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| Abstract: | A method for direct measurements of the binding of N-acetyl glucosamine polymers to the catalytic site of lysozyme is described. The method utilizes the dye Biebrich scarlet for which the enzyme has a single binding site with a dissociation constant of 0.3 mM. The data indicate that the trimer and hexamer of N-acetyl glucosamine do not displace the dye from the unproductive binding site of lysozyme to which they both bind with a dissociation constant of about 10 μM. The two sugars competitively displace the dye from the catalytic site of the enzyme, allowing one to differentiate between productive and unproductive binding of these two substrates. Spectrophotometric determinations of the concentrations of the enzyme-dye complex in the presence of various concentrations of substrate gave productive binding dissociation constants for the trimer and hexamer of 20 mM and 5 μM respectively. |
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