Mechanism of Lysozyme Catalysis: Role of Ground-State Strain in Subsite D in Hen Egg-White and Human Lysozymes

Melvin Schindler, Nathan Sharon, Yehudah Assaf, David M. Chipman

Research output: Contribution to journalArticlepeer-review

49 Scopus citations


The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-β(1 → 4)-MurNAc-β(1 → 4)-GlcNAc-β(1 → 4)-GlcNAc, GlcNAc-β(1 → 4)-MurNAc-β(1 → 4)-GlcNAc-β(1 → 4)-N-acetyl-d-xylosamine, and GlcNAc-β(1 → 4)-GlcNAc-β(1 → 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-d-glucosamine, or N-acetyl-d-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-d-glucosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformations of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-β(1 → 4)-MurNAc-β(1 → 4)-GlcNAc-β(1 → 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is “looser” than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.

Original languageEnglish
Pages (from-to)423-431
Number of pages9
Issue number3
StatePublished - 1 Feb 1977

ASJC Scopus subject areas

  • Biochemistry


Dive into the research topics of 'Mechanism of Lysozyme Catalysis: Role of Ground-State Strain in Subsite D in Hen Egg-White and Human Lysozymes'. Together they form a unique fingerprint.

Cite this