From Proteopedia

Revision as of 04:47, 30 October 2010

Lysozyme

Lysozyme is an important enzyme that is commonly found in vertebrate cells and secretions capable of destroying cell walls.

Activity

Lysozyme is an enzyme effective in its catalytic degradation of the peptidoglycan in bacterial cell walls, specifically by catalyzing the hydrolysis of 1,4 beta linkages of the residues, N-acetylmuramic acid and N-acetyl-D-glucosamine in cell wall peptidoglycan. This enzyme similarly catalyzes hydrolysis of 1,4 beta linkages of poly N-acetylglucosamine residues of chitin, a major constituent of fungi cell walls as well as exoskeletons such as those of insects and crustaceans.

Occurrance

This cell wall degradation often leads to cell death, displaying the bactericidal function of lysozyme which is a main function of lysozyme. The effectiveness of lysozyme in degrading cells walls, especially those of gram-positive bacterial cells is a function that naturally leads to the incorporation of the lysozyme enzyme into organisms as a bactericidal agent. Lysozyme is readily present in the living cells and mucosal secretions of vertebrates including tears and saliva.

History

Initially in 1909 Laschtschenko described lysozyme which he found displaying antibacterial effects in a chicken egg. Alexander Fleming then observed the antibacterial lysing action of lysozyme by treating cells with mucus, and developed the name lysozyme. David Phillips solved the structure of lysozyme using X-ray diffraction methods, lysozyme being only the second protein and the first enzyme to have been structurally determined using these X ray diffraction procedures. Phillips determined the full sequence of lysozyme as well as the enzyme structure as well as the mechanism, which performed the catalytic process of lysozyme. This mechanism provided insight into the influence of structure on the catalyzing action of lysozyme and enzymes as a whole.

Structure

The presence of lysozyme in hen egg whites has led to this particular lysozyme, Hen egg white lysozyme, to prevail as the most heavily studied lysozyme species. This increased understanding of the mechanism of lysozyme allows a more specific analysis of lysozyme structure and specific function. Visualize the overall structure here

Hen egg white lysozyme is a smaller ellipsoidal enzyme composed of 129 amino acid residues leading to a single polypeptide chain of 14.3 kD. The amino acid polypeptide sequence of 129 amino acid residues is responsible for the secondary and tertiary structure of the lysozyme molecule. HERE. This polypeptide backbone can be mapped from amino to carboxyl end, mapping the path of the polypeptide after proper folding has occurred HERE.

The backbone of this 129 amino acid enzyme conforms to a very specific and ordered molecule with the help of the secondary structural interactions. Lysozyme contains five alpha helical regions and five regions containing beta sheets. Linking these secondary structures, a number of beta turns and large amount of random coil makes up the remainder of the polypeptide backbone. The polypeptide backbone of lysozyme involved in the 3 antiparallel beta sheets display the beta hairpin motif of supersecondary structure. The increased stability of the antiparallel beta sheet due to the proper alignment of hydrogen bonds between sheets allows for the presence of the Beta hairpin motif.

Hydrogen Bonding

The hydrogen bonding patterns in secondary structure have an important role in determining protein structure. Limited by the various torsion angles of the alpha carbon-nitrogen and alpha carbon-carbon bonds in each residue, each secondary structure (alpha helices and beta sheets) display specific patterns of hydrogen bonding of the amino acid residues. Outlines the various hydrogen bonding interactions between the various hydrogen bond donators and acceptors.

Disulfide Bonding

The conformation of the lysozyme peptide is affected by the presence of 4 disulfide bridges between 8 Cystein residues occurring within the peptide backbone. The presence of these disulfide bridges assist in the folding, stability of the structure, and overall function of lysozyme.

Amino Acid Residues

The amino acids present in the lysozyme polypeptide sequence have a direct influence not only on primary structure, but the following secondary and tertiary structural changes which can be influence by polarity and charge. The various amino acid residues differ in their properties because of the great variety of side chains present on each amino acid. Polar and nonpolar, and charged and uncharged side chains lead to various degrees of hydrophobicity and hydrophilicity which can have a very dominant effect on protein folding. In lysozyme, these side chains are displayed for each amino acid residue.

Polarity

The nature of the amino acid sidechains in the lysozyme polypeptide sequence leads to regions of varying hydrophobic natures and polarities of the enzyme structure. The presence of certain regions of hydrophilicity and hydrophobicity is a driving force in determining protein structure when folding. The varying polarities of the side chains influence the locations of residues in the enzyme structure. Nonpolar residues will display hydrophobic tendencies occurring mostly on the interior of the enzyme while polar residues will increase in abundance on the surface of the protein in order to contact the aqueous solvent satisfying their hydrophilic nature. By observing the following structural depictions of lysozyme with polar molecules colored and nonpolar molecules colored the influence of polarity on nucleotide arrangement and protein folding is evident. The presence of water molecules interacting with thee various hydrophilic residues is depicted to further display how polarity affects structure.

Charge

Charges of the various regions of the lysozyme structure display a hydrophilic nature and thus also affect the location of that region of polypeptides and folding of the protein. Charged regions of the protein will display hydrophilic tendencies and therefore be located on the surface of the lysozyme molecule where they can interact with the aqueous solvent. Non-charged portions will display hydrophobic tendencies and be located on the interior of the molecule. This effect can be visualized with charged molecules represented by cationic and anionic, and uncharged regions colored in

Active Site

Enzymes contain a mechanism of action that utilizes the specificity of binding to a substrate to produce an enzyme substrate complex, which can then proceed with the mechanism. The active site has specific residues that facilitate the binding of the substrate through a specific three-dimensional arrangement facilitating the substrate binding, prior to catalyzing the reaction and releasing products. Lysozyme contains a cleft in its structural arrangement, which allows for the location of the substrate-binding site. The active site of lysozyme is arranged to accommodate an oligosaccharide substrate size of six residues. Along with this geometric complementing of the active site, the sidechains of Glu 35 and Asp 52, which are considered lysozyme’s active site residues, assist in the catalysis of the glycosidic bond hydrolysis. In order to accomplish the acetyl hydrolysis that occurs, the transiently formed oxonium ion produced by the protination of the oxygen atom and the subsequent cleavage of the C-O bond. Lysozyme mediates this reaction, and the Glu 35 and Asp 52 residues serve to perform general acid catalysis and stabilize the ion transition state respectively to yield a product.

Ligand

The presence of a ligand on the lysozyme molecule would display a smaller molecule bound to the surface of the lysozyme molecule. Ligands can biologically assist catalyze reactions. Enzyme substrates can be viewed as ligands that directly bind to the active site of the enzyme. In the case of the lysozyme mechanism, the oligosaccharide consisting of 6 substrate residues would bind to the active site of the lysozyme molecule to begin the reaction. The presence of a ligand clearly distinguished from the enzyme components is visible in this model.

Mechanism

Lysozyme catalyzes a reaction, specifically the hydrolysis of a glycoside. Lysozyme will attach to a bacterial cell wall through the binding to the hexasaccharide substrate. The D residue of this oligosaccharide in order to properly fit in the active site must be distorted to the half chair conformation. Following the transfer of Glu 35 proton to the O1 atom between the D and E sugar rings, The cleavage of the C1-O1 bond forms the positively charged oxonium ion. The Asp 52 carboxylate group performs electrostatic catalysis to stabilize the presence of this transition state. The nucleophilic attack of the Asp 52 carboxylate group to the C1 of the D ring forms a covalent intermediate. Water replaces the E sugar ring. Glu 35 performs general base catalysis to assist in hydrolyzing the covalent bond releasing the D ring product following another oxonium ion transition state completing the catalytic cycle.

The mechanism proceeds as follows:

Glu 35 acts as a general acid catalyst and a general base catalyst, and Asp 52 acts as a covalent catalyst, to help mediate the reaction with a significant increase in rate of hydrolysis of the substrate than in an uncatalyzed equivalent reaction.