Introduction to Molecular Biology: From Cells to Atoms - Anthony Rees, Michael Sternberg 2002

Proteins
Proteins as enzymes: lysozyme

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Fig. 14.1.

Lysozyme is an enzyme capable of destroying certain bacterial Cells by cleaving the polysaccharide chains of The Cell wall. Deprived of its rigid cell wall, the bacterium bursts under the osmotic Shock caused by the rapid influx of Water into The Cell.

The cell wall polysaccharide is a polymer consisting of alternating residues of Two Types of sugars: N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). These sugars, which possess a ß-configuration relative to the anomeric C₁ ^-carbon atom, form a polymer chain via glycosidic bonds between the C₁ carbon atom of one sugar ring and the C₄ carbon atom of the next (Ch. 31).

Structure of lysozyme. Hen egg-white lysozyme consists of a single polypeptide chain comprising 129 residues and containing four disulfide bridges (Ch. 10). The enzyme can bind

The polysaccharide is cleaved via the Hydrolysis of the glycosidic bond between the sugar rings located in sites D and E. Crystallographic Data analysis suggests that immediately before and during hydrolysis, the sugar ring in site D adopts not a standard chair conformation, but a half-chair conformation, characterized by the fact that five of the six atoms forming the sugar ring lie almost in a single plane (Fig. 14.3).

Glutamic acid 35 and aspartic acid 52 play a central role in lysozyme function. The side chains of these residues are located close to the glycosidic bond (approximately 0.3 nm away) between the sugar rings localized in sites D and E. Glu 35 resides in a nonpolar environment, which allows its carboxyl group to remain protonated (i.e., with inhibitors chemically similar to the cell wall polysaccharide. Using X-Ray Diffraction Analysis, Phillips and coworkers determined the three-dimensional structures of both the native protein and the protein co-crystallized with an inhibitor. They found that lysozyme consists of two domains forming a cleft that houses the Active Site, which is capable of binding a hexasaccharide; each of the six sugar rings binds to a specific subsite on the enzyme, designated as sites A, B, C, D, E, and F (Fig. 14.2). in the —COOH form). In contrast, the environment of Asp 52 is polar, so the carboxyl group of this residue is deprotonated (i.e., in the —COO⁻ form).

Fig. 14.2. STRUCTURE OF THE lysozyme molecule in the active site region. The substrate (NAG-NAM)₃ bound in the active site is shown in brown. NH and CO groups are highlighted in gray and black, respectively. Hydrogen Bonds are represented by dashed straight lines. Note the close proximity of the rings of two Tryptophan residues, Trp 62 and Trp 63, to the sugars located in sites A and B. Van der Waals contacts and hydrogen bonding with these residues provide additional stabilization of the substrate within the active site.

Fig. 14.3.

The hydrolysis reaction can be divided into several stages.

1. The —COOH group of the Glu 35 residue donates its proton to the glycosidic oxygen, resulting in the Cleavage of the bond between this oxygen atom and the C₁ carbon atom of the sugar ring located in site D. The resulting fragment of the initial polysaccharide, which includes the sugar rings located in sites E and F, is a product and can dissociate from the enzyme-substrate complex.

2. The sugar ring in site D adopts a distorted conformation corresponding to the Transition State. In this state, the carbon atom becomes positively charged. A carbon atom in such a state is referred to as a carbonium ion (carbocation). It is stabilized by the negative charge of the nearby Asp 52 residue.

3. A hydroxyl ion (OH⁻), provided by an ambient water molecule, attacks the carbonium ion, thereby converting the second fragment of the cleaved polysaccharide into a reaction product. Simultaneously, upon binding a hydrogen ion (H⁺), the carboxyl group of Glu 35 becomes protonated, reverting to the —COOH form.

4. The enzyme has now returned to its initial state and is ready to catalyze the cleavage of another polysaccharide molecule.

This example illustrates several General Principles of Enzymatic Catalysis:

1. Increase in substrate energy due to the structural distortion of the NAM sugar ring located in site D.

2. The unique microenvironment of Glu 35, which facilitates the generation of a reactive proton.

3. Proper orientation of the proton in Glu 35, which is essential for attacking the glycosidic bond.

4. Reduction of the Free energy of the transition state through the stabilization of the carbonium ion by the carboxyl group of the Asp 52 residue.