Question

The lysozyme reaction is shown below:

1. The lysozyme reaction is a bimolecular enzyme-catalyzed reaction. The two substrates are the NAG-NAM polymer and a water molecule. What bimolecular kinetic model will lysozyme follow? Justify your answer.

Glu-35 Glu-35 H00 HO НО Lo—NAG HO— HO-NAG + H20_ NAG. NAG 0 0 | NHAC NHAC 0 - A NHAC NHAC 0 0 0 Asp-52 Asp-52 COO- COO Note: NAG stands for N-acetylglucosamine. The monomer explicitly drawn is N-acteylmuramic acid (NAM) and is connected by oxygens into a polymer of alternating NAG-NAM subunits.

2. Typically, we would confirm the bimolecular model with a set of kinetic experiments and the resulting Lineweaver-Burk plots. Explain why it would be experimentally difficult to use this strategy for lysozyme.

Answer 1

The most abundant and diverse prokaryote microorganisms in our planet are bacteria, which are surrounded by a sophisticated and complex structure called bacterial cell wall. This structure is important because it not only defines the bacterial cell shape, but it also provides protection in hostile and very often unpredictable environments, both in biological and nonbiological systems 1.

The main component of bacterial cell wall is peptidoglycan, which is composed of alternating units of N‐acetylglucosamine (NAG) and N‐acetylmuramic acid (NAM), linked by β‐(1,4) bonds 2.

Gram‐positive bacteria have a single lipid membrane surrounded by a cell wall composed of a thick layer of peptidoglycan, while in Gram‐negative bacteria the cell wall consists of a thin layer of peptidoglycan placed between the cytoplasmic and outer lipid membranes (Fig. 1A). There has been an indefatigable search throughout the years for different strategies to disrupt the bacterial cell wall to fight against invasion of pathogenic bacteria.

Figure 1

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(A) Structural comparison between the bacterial cell wall of characteristic Gram‐positive and Gram‐negative microorganisms. (B) Upper; Specific cleavage sites on the peptidoglycan chain for lysozyme, which catalyzes the hydrolysis of the β‐1,4 bond between N‐acetylmuramic acid and N‐acetylglucosamine Bottom; Mechanism of action of a lysozyme. In the enzyme‐substrate complex, the Glu‐35 residue donates a proton (red) to the glycosidic bond, generating an oxonium that is stabilized by Asp‐52 via a covalent bond. The enzyme‐substrate intermediate complex reacts with a water molecule (light blue), where Glu‐35 accepts a proton from the product of hydrolysis and leaves the enzyme unchanged. References: R= cell wall oligosaccharide chain, R'= cell wall peptide side chain. (C) Progressive effect of lysozyme on the peptidoglycan of bacteria and lytic activity.

Lysozyme is an enzyme that cleaves peptidoglycan in bacterial cell walls by catalyzing the hydrolysis of β‐(1,4) linkages between the NAM and NAG saccharides

In this enzymatic reaction, the residue Glu‐35 acts as an acid, donating a proton to the glycosidic bond, generating an oxonium that is then stabilized by a covalent bond with Asp‐52. The addition of a water molecule completes the hydrolysis and regenerates the protonated form of Glu‐35 .