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.
Highlights
From the *Departmentof Microbiology and Immunology and the TDepartmentof Biochemistry, Tempk University School of Medicine,Philadelphia, Pennsylvania19140
Doglycan chains producedby the same organism in the2 for arecent review),and enzymesthat hydrolyze the peptide, presence of benzylpenicillin was characterized as a amide, and glycosidic bonds in wall peptidoglycans have been processivexodisaccharidaseS.pecifilcabelso,ne ([“CIGal) added to the nonreducing enodfschains, and the other (3H from [‘H]N&H4) incorporated into the reducing endsof the chains, were used to establish that an enzyme molecule binds at the nonreducing terminus described [1,2,3]
Reaction rates increased with the lengthof the strates, such as walls themselves, or insoluble peptidoglycan polymer chain to give a maximum of 9 1bonds cleaved/ residues; (ii) low-molecular-weight peptidoglycan fragments polymeric substrate
Summary
Autolytic peptidoglycan hydrolases (autolysins) are endog- tain glycan strands of approximately uniform length that are enous bacterial enzymes that hydrolyze specific bonds within fully substituted with peptides andthus consist of linear insoluble cell walls, thereby causing the walls to lose their chains of DSP, units. The s-peptido- Separation and Identification of Products of Enzymatic Hyglycans are soluble, linear, uncross-linked chains consisting of 44-47 8,l-l-linked DSP, units with MurNAc at the reducing terminus These s-peptidoglycan chains are secreted into the medium by benzylpenicillin-inhibited cells [13].
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