The mechanism of soluble peptidoglycan hydrolysis by an autolytic muramidase. A processive exodisaccharidase

J. F. Barrett, D. L. Dolinger, V. L. Schramm, G. D. Shockman

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12 Scopus citations


The action of purified N-acetylmuramoylhydrolase (muramidase, EC of Streptococcus faecium ATCC 9790 on linear, uncross-linked, soluble, peptidoglycan chains produced by the same organism in the presence of benzylpenicillin was characterized as a processive exodisaccharidase. Specific labels, one ([14C]Gal) added to the nonreducing ends of chains, and the other (3H)NaBH4) incorporated into the reducing ends of the chains, were used to establish that an enzyme molecule binds at the nonreducing terminus and sequentially hydrolyzes the glycosidic bonds, releasing disaccharide-peptide units. An enzyme molecule remains bound to a chain, and is not released at a detectable rate, until hydrolysis of that chain is complete. Reaction rates increased with the length of the polymer chain to give a maximum of 91 bonds cleaved/min/enzyme molecule for hydrolysis of a continuous polymeric substrate. The relationship between hydrolytic rate and glycan chain length is consistent with hydrolysis of bonds with the chain followed by slow release of enzyme from the distal, reducing terminus. This mechanism was experimentally confirmed by analysis of product formation during hydrolysis with stoichiometric mixtures of enzyme and soluble peptidoglycan chains. Kinetic analyses showed an apparent K(m) of 0.17 μM for the enzyme, independent of substrate polymer length. The dissociation constant for the initial enzyme-substrate complex was calculated to be 1.5 nM. Kinetic analyses are consistent with one catalytic site per enzyme molecule. The k(cat)/K(m) value of 9 x 106 M-1 s-1 is near the limit imposed by diffusion for the initial hydrolytic events when long chains are hydrolyzed. The kinetic and physical properties of this muramidase are highly consistent with its location outside of the cellular permeability barrier and its ability to remain with and hydrolyze appropriate bonds in the cell wall in such an environment.

Original languageEnglish (US)
Pages (from-to)11818-11827
Number of pages10
JournalJournal of Biological Chemistry
Issue number19
StatePublished - 1984
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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