The mechanism of soluble peptidoglycan hydrolysis by an autolytic muramidase. A processive exodisaccharidase
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Abstract:
The action of purified N-acetylmuramoylhydrolase (muramidase, EC 3.2.1.17) 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 from [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 bond 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 within 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 Km of 0.17 microM 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 Kcat/Km value of 9 X 10(6) 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.
Insights
Streptococcus faecium
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- N-acetylmuramoylhydrolase (muramidase) is an enzyme crucial for bacterial cell wall metabolism.
- Understanding its action mechanism is key to developing novel antibacterial strategies.
Purpose of the Study:
- To characterize the action of purified N-acetylmuramoylhydrolase from Streptococcus faecium.
- To elucidate the enzyme's mechanism as a processive exodisaccharidase.
Main Methods:
- Utilized radiolabeled peptidoglycan chains (nonreducing and reducing ends) to track enzyme activity.
- Performed kinetic analyses to determine enzyme-substrate interactions and catalytic efficiency.
- Investigated enzyme-substrate binding and release dynamics.
Main Results:
- The enzyme acts as a processive exodisaccharidase, releasing disaccharide-peptide units sequentially from the nonreducing end.
- Enzyme remains bound to the substrate until hydrolysis is complete, with reaction rates increasing with polymer length.
- Apparent Km was 0.17 microM, dissociation constant was 1.5 nM, indicating high-affinity binding.
- Kcat/Km value suggests near-diffusion-limited catalytic efficiency for initial hydrolytic events.
Conclusions:
- The muramidase exhibits properties consistent with an extracellular location, enabling efficient cell wall hydrolysis.
- Its processive action and high catalytic efficiency highlight its significance in Streptococcus faecium cell wall turnover.
- Findings provide insights into enzyme-substrate interactions and potential targets for antimicrobial development.