Human lysozyme-catalyzed reaction of chitooligosaccharides

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Human lysozyme and hen egg-white lysozyme are enzymes that catalyze the cleavage of glycosidic linkages.
• Subsite D's binding free energy is crucial for understanding lysozyme's enzymatic mechanism.
• Previous studies suggested negative binding free energy for subsite D in human lysozyme.

Purpose of the Study:

• To compare the enzymatic reaction time-courses of human lysozyme and hen egg-white lysozyme with chitopentaose.
• To investigate the role of subsite D's binding free energy in human lysozyme's catalytic activity.
• To reconcile discrepancies between theoretical models and experimental data for human lysozyme.

Main Methods:

• High-performance gel-filtration chromatography was used to measure reaction time-courses.
• Kinetic parameters, including rate constants for cleavage and transglycosylation, were determined.
• Computational modeling was employed to simulate reaction time-courses based on different binding free energy assumptions.

Main Results:

• Human lysozyme exhibited significantly higher rate constants for glycosidic bond cleavage and transglycosylation compared to hen egg-white lysozyme.
• Experimentally determined time-courses did not align with simulations assuming negative binding free energy at subsite D in human lysozyme.
• Variations in rate constants did not resolve the discrepancy, suggesting an issue with the initial assumption.

Conclusions:

• The binding free energy of subsite D in human lysozyme is likely positive, similar to hen egg-white lysozyme, rather than negative.
• The previously reported negative binding free energy for human lysozyme's subsite D may be inaccurate.
• This finding necessitates a re-evaluation of the catalytic mechanism of human lysozyme.