Lysozyme

It is a glycoside hydrolase that catalyzes the following process:

Hydrolysis of (1→4)-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins (dextrins derived from chitin)

Peptidoglycan is the major component of gram-positive bacterial cell wall. This hydrolysis in turn compromises the integrity of bacterial cell walls causing lysis of the bacteria.

Lysozyme is abundant in secretions including tears, saliva, human milk, and mucus. It is also present in cytoplasmic granules of the macrophages and the polymorphonuclear neutrophils (PMNs). Large amounts of lysozyme can be found in egg white. C-type lysozymes are closely related to α-lactalbumin in sequence and structure, making them part of the same glycoside hydrolase family 22. In humans, the C-type lysozyme enzyme is encoded by the LYZ gene.

Hen egg white lysozyme is thermally stable, with a melting point reaching up to 72 °C at pH 5.0. However, lysozyme in human milk loses activity very quickly at that temperature. Hen egg white lysozyme maintains its activity in a large range of pH (6–9). Its isoelectric point is 11.35. The isoelectric point of human milk lysozyme is 10.5–11.

Function and mechanism

The enzyme functions by hydrolyzing glycosidic bonds in peptidoglycans. The enzyme can also break glycosidic bonds in chitin, although not as effectively as true chitinases.

Lysozyme's active site binds the peptidoglycan molecule in the prominent cleft between its two domains. It attacks peptidoglycans (found in the cell walls of bacteria, especially Gram-positive bacteria), its natural substrate, between N-acetylmuramic acid (NAM) and the fourth carbon atom of N-acetylglucosamine (NAG).

Shorter saccharides like tetrasaccharide have also shown to be viable substrates but via an intermediate with a longer chain. Chitin has also been shown to be a viable lysozyme substrate. Artificial substrates have also been developed and used in lysozyme.

Mechanism

Phillips

The Phillips mechanism proposed that the enzyme's catalytic power came from both steric strain on the bound substrate and electrostatic stabilization of an oxo-carbenium intermediate. From X-ray crystallographic data, Phillips proposed the active site of the enzyme, where a hexasaccharide binds. The lysozyme distorts the fourth sugar (in the D or -1 subsite) in the hexasaccharide into a half-chair conformation. In this stressed state, the glycosidic bond is more easily broken. An ionic intermediate containing an oxo-carbenium is created as a result of the glycosidic bond breaking. Thus distortion causing the substrate molecule to adopt a strained conformation similar to that of the transition state will lower the energy barrier of the reaction.

The proposed oxo-carbonium intermediate was speculated to be electrostatically stabilized by aspartate and glutamate residues in the active site by Arieh Warshel in 1978. The electrostatic stabilization argument was based on comparison to bulk water, the reorientation of water dipoles can cancel out the stabilizing energy of charge interaction. In Warshel's model, the enzyme acts as a super-solvent, which fixes the orientation of ion pairs and provides super-solvation (very good stabilization of ion pairs), and especially lower the energy when two ions are close to each other.

The rate-determining step (RDS) in this mechanism is related to formation of the oxo-carbenium intermediate. There were some contradictory results to indicate the exact RDS. By tracing the formation of product (p-nitrophenol), it was discovered that the RDS can change over different temperatures, which was a reason for those contradictory results. At a higher temperature the RDS is formation of glycosyl enzyme intermediate and at a lower temperature the breakdown of that intermediate.

Covalent mechanism

In an early debate in 1969, Dahlquist proposed a covalent mechanism for lysozyme based on kinetic isotope effect, but for a long time the ionic mechanism was more accepted. In 2001, a revised mechanism was proposed by Vocadlo via a covalent but not ionic intermediate. Evidence from ESI-MS analysis indicated a covalent intermediate. A 2-fluoro substituted substrate was used to lower the reaction rate and accumulate an intermediate for characterization. The amino acid side-chains glutamic acid 35 (Glu35) and aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, whereas Asp52 acts as a nucleophile to generate a glycosyl enzyme intermediate. The Glu35 reacts with water to form hydroxyl ion, a stronger nucleophile than water, which then attacks the glycosyl enzyme intermediate, to give the product of hydrolysis and leaving the enzyme unchanged. This type of covalent mechanism for enzyme catalysis was first proposed by Koshland.

More recently, quantum mechanics/ molecular mechanics (QM/MM) molecular dynamics simulations have been using the crystal of HEWL and predict the existence of a covalent intermediate. Evidence for the ESI-MS and X-ray structures indicate the existence of covalent intermediate, but primarily rely on using a less active mutant or non-native substrate. Thus, QM/MM molecular dynamics provides the unique ability to directly investigate the mechanism of wild-type HEWL and native substrate. The calculations revealed that the covalent intermediate from the covalent mechanism is ~30 kcal/mol more stable than the ionic intermediate from the Phillips mechanism. These calculations demonstrate that the ionic intermediate is extremely energetically unfavorable and the covalent intermediates observed from experiments using less active mutant or non-native substrates provide useful insight into the mechanism of wild-type HEWL.

Inhibition

Imidazole derivatives can form a charge-transfer complex with some residues (in or outside active center) to achieve a competitive inhibition of lysozyme. In Gram-negative bacteria, the lipopolysaccharide acts as a non-competitive inhibitor by highly favored binding with lysozyme.

Non-enzymatic action

Despite that the muramidase activity of lysozyme has been supposed to play the key role for its antibacterial properties, evidence of its non-enzymatic action was also reported. For example, blocking the catalytic activity of lysozyme by mutation of critical amino acid in the active site (52-Asp -> 52-Ser) does not eliminate its antimicrobial activity. The lectin-like ability of lysozyme to recognize bacterial carbohydrate antigen without lytic activity was reported for tetrasaccharide related to lipopolysaccharide of Klebsiella pneumoniae. Also, lysozyme interacts with antibodies and T-cell receptors.

Enzyme conformation changes

Lysozyme exhibits two conformations: an open active state and a closed inactive state. The catalytic relevance was examined with single walled carbon nanotubes (SWCN) field effect transistors (FETs), where a singular lysozyme was bound to the SWCN FET. Electronically monitoring the lysozyme showed two conformations, an open active site and a closed inactive site. In its active state lysozyme is able to processively hydrolyze its substrate, breaking on average 100 bonds at a rate of 15 per second. In order to bind a new substrate and move from the closed inactive state to the open active state requires two conformation step changes, while inactivation requires one step.

Superfamily

The conventional C-type lysozyme is part of a larger group of structurally and mechanistically related enzymes termed the lysozyme superfamily. This family unites GH22 C-type ("chicken") lysozymes with plant chitinase GH19, G-type ("goose") lysozyme GH23, V-type ("viral") lysozyme GH24 and the chitosanase GH46 families. The lysozyme-type nomenclature only reflects the source a type is originally isolated from and does not fully reflect the taxonomic distribution. For example, humans and many other mammals have two G-type lysozyme genes, LYG1 and LYG2.