Lysozyme Lecture Notes
Enzymatic Cleavage of Glycosides: Lysozyme
Lysozyme, also known as muramidase, cleaves N-acetylmuramide linkages and is found in secretions like tear fluid and saliva. Eels produce mucus containing lysozyme for protection against bacterial infections.
Lysozyme's Role in Defense
Lysozyme acts as a first line of defense against bacterial infections, especially those with peptidoglycan cell walls. Alexander Fleming discovered its antibiotic activity, in addition to penicillin. It prevents bacterial infections like conjunctivitis and is part of our innate immune response.
Lysozyme in Eggs
Eggs contain a significant amount of lysozyme, crucial for protecting the developing embryo from bacterial infections. Hen egg white lysozyme is extracted from eggs and used as a preservative in the food industry.
Lysozyme Family
Originally thought to be a small group, lysozyme is now recognized as part of a larger family of enzymes resulting from gene duplication events. Lactalbumin, similar in structure and sequence to lysozyme, is involved in lactose synthesis and has antibacterial properties in milk. Recently, mammalian testicular versions of lysozyme-like enzymes have been discovered. Lactalbumin is a calcium-binding protein, but hen egg white lysozyme, which is the primary focus of this lecture, does not bind calcium.
Lysozyme and Peptidoglycan
Lysozyme destroys bacterial cell walls by hydrolyzing beta 1-4 glycosidic linkages between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), the major components of peptidoglycan cell walls. Lysozyme was the first enzyme crystallized, making it crucial in understanding the structure and mechanism of glycosyl hydrolases.
Peptidoglycan Structure
Peptidoglycan monomers consist of polymers of NAM and NAG, linked by peptides that uniquely use D-amino acids rather than L-amino acids. Peptidoglycans are present in gram-positive bacteria as a thick outer wall and in gram-negative bacteria between the inner and outer membranes. Lysozyme can break down both. Gram-positive bacteria have multiple peptidoglycan layers composed of alternating NAM and NAG copolymers cross-linked with a pentapeptide, while gram-negative bacteria have a thinner, single peptidoglycan layer.
Mechanism Discussion
Lysozyme cleaves the NAM-NAG copolymer using water to hydrolyze the linkages.
Retaining vs. Inverting Mechanisms
Lysozyme employs a retaining mechanism, similar to Golgi alpha-mannosidase. In inverting mechanisms, the oxygen position changes, while retaining mechanisms maintain the oxygen position, forming a covalent intermediate.
Koshland Mechanism
The Koshland mechanism, proposed in 1953, suggests the formation of a covalent intermediate between the nucleophile and the sugar during catalysis.
David Phillips and Lysozyme Structure
In 1965, David Phillips determined the three-dimensional structure of lysozyme, the first enzyme structure ever solved. The structure, primarily alpha-helical with a clasp-like shape, revealed a canyon-like active site with a negative charge, suggesting it can hold a polymer of sugars. Phillips modeled a six NAM-NAG copolymer into the active site, noting that the conformation of one sugar had to be altered to fit.
Active Site Features
The active site contains aspartate and glutamic acid, considered catalytic amino acids. Like other glycosyl hydrolases, lysozyme has amino acids that hold the sugar in place, such as tryptophan. Cleavage occurs between sugars D and E.
Substrate Modeling and Conformational Change
Phillips observed that sugar D's conformation had to change from a chair to a half-chair to fit into the active site, suggesting a transition state shave of the sugar before cleavage could take place. This conformational change involves the oxygen popping down from the chair conformation to a half-chair conformation, making it easier to cleave the sugar. Glutamic acid donates a proton, and the sugar is held in an oxocarbenium state until water comes in.
Phillips Mechanism
Proton Donation: Glutamic acid donates a proton.
Leaving Group Departure: The group leaves, and the aspartate holds the sugar in position in an oxocarbenium ion state.
Water Attack: Water comes in and re-protonates the glutamic acid, attacking the oxocarbenium ion, retaining the hydroxyl position.
Issues with the Phillips Mechanism
While other retaining glycosyl hydrolases followed the Koshland mechanism, textbooks adopted the Phillips mechanism. Steve Withers later disproved this mechanism using fluoroglycosides.
Withers' Experiment and Proof of Covalent Intermediate
Withers used fluoroglycosides to trap the covalent intermediate and mutated glutamic acid to glutamine to remove the general acid-base. Mass spectrometry showed that the molecular mass of hen egg white lysozyme increased when glutamic acid was changed to glutamine, indicating a trapped covalent intermediate with the aspartate. This experiment demonstrated that the covalent intermediate exists and the long-lived oxocarbenium ion does not.
Revised Understanding
The widely-accepted Phillips mechanism was incorrect and the Koshland mechanism is correct. Fluorinated sugars trap the nucleophile in a covalent intermediate state, implying that black cell hydrolases with a retaining mechanism all work via a covalent intermediate. By deactivating the general acid base a covalent intermediate is formed, indicating that normal sugars, not just fluorinated ones yield a covalent intermediate in the reaction.
Common Mechanisms in Glycosylhydrolases
Glycosylhydrolases share similar mechanisms, including a covalent intermediate and similar active sites. Substrate stabilization occurs before cleavage, sometimes requiring sugar distortion. Tryptophan helps stabilize and position the sugar for cleavage. These enzymes also have similarly charged surfaces and conserved active sites.
Convergent Evolution
Despite different overall structures when comparing mannosidases from the ER, Golgi to lysozyme the active site is largely the same.
That is a good example of convergent evolution in glycosylhydrolases where similar active sites and mechanisms are developed independently.