Human Lysozyme
created by Sydney M. Strader
Human lysozyme (PDB ID = 1RE2) is an enzyme that catalyzes the hydrolysis of beta-1,4 glycosidic linkages in the peptidoglycan of bacterial cell walls. In other cases lysozyme has been linked to inactivation of viruses, surveillance of membranes, stimulation of monocytes, and antitumor activity. It is present predominately in human tears and saliva (2,7). Lysozyme was considered as a potential antibiotic before penicillin was discovered. The enzyme is small for a globular protein, but it is still too large to travel through cell membranes. In nature, it exists as an extracellular protein. Due to this, it is not particularly useful as a drug. In addition, drugs do not commonly target lysozyme because there is no reason to inhibit lysozyme activity when it defends against bacterial infection (13).
Human lysozyme consists of one polypeptide chain of 130 residues, with a molecular weight of 14,700.67 Da. The physiological isoelectric point is 9.28 (12). The enzyme has four alpha helices, four random coils, and a double antiparallel beta-pleated sheet. The four disulfide bonds (connecting Cys-65 to Cys-81, Cys-77 to Cys-95, Cys-30 to Cys-116, andCys-6 to Cys128) link three of the alpha helices and a random coil to intrinsically unfolded loops (6). 36% of the amino acids that make up the polypeptide chain are hydrophobic, most of these being tucked away on the inside of the protein or in the catalytic cleft that divides the two domains.
The helix-loop-helix domain (HLH) from Asp-87 to Arg-114 is particularly catalytically important because it forms the upper lip of the cleft that binds the ligands. It is within the region with the greatest degree of conservation across similar proteins. Sixteen of the twenty eight residues are identical and five of the replaced residues are chemically conservative.
The cleft can accommodate six polysaccharide sugars at one time. The sugars that lysozyme binds to come from peptidoglycan, the macromolecule that makes up the cell walls of bacteria. Lysozyme catalyzes the breaking of the beta-1,4 linkages between N-acetylglucosamine and N-acetylmuramic acid, disrupting the cell wall, and killing the bacteria (10). Of the six monosaccharides (consider them monosaccharides A through F) that bind to the active site, hydrolysis of the glycosidic bond happens between those represented as D and E. D is strained into a conformation similar to a transition state. Glu-35 transfers a proton to the oxygen of the glycosidic bond and the C-O bond is cleaved. Polysaccharide residues D and E are no longer bonded, and the E-linked half of the polysaccharide is released, but Glu-35 now has a negatively charged oxygen and D is an unstable, positively charged ion (Figure A-b). Asp-53 has a negatively charged oxygen as well, and through resonance, stabilizes the positively charged carbon. A water molecule from the solvent can then protonate Glu-35, and the resulting hydroxide can bond with residue D, neutralizing the charge and causing its release from the cleft (8). At this stage, the enzyme is also prepared to catalyze another reaction.
Glu-35 and Asp-53 are the critical to enzymatic activity. When Glu-35 is replaced with Gln-35 (the only change being that the hydroxyl group is replaced with an amine), the enzyme can still bind the substrate but there is no catalytic activity and the glycosidic linkages are not hydrolyzed. This is due to the fact that there is no longer a hydroxyl group to donate a hydrogen to the bond (the hydrolysis process). When Asp-53 is changed to Asn-53 (the negatively charged oxygen changed to an amine), the substrate is still bound, and there is catalytic activity, but it is only 5% of the activity of an Asp-53 lysozyme. Hydrolysis can still be carried out but it is much more unfavorable now that the unstable intermediate is no longer stabilized by resonance with the negatively charged oxygen of Asp-53 (8). The enzymatic activity of lysozyme is possible because of the slight flexing and conformational changes that the protein can accommodate. This is due to small pockets in between amino acids that allow the molecule to flex and bend around the substrate.
Hen egg-white lysozyme is very similar in structure to human lysozyme. The Z score is 25.5 (1) and the E-score is 4e-53 (9). A high Z score (above 2) and a low E score (below 0.05) both indicate protein similarity. The Z score comes from the Dali server and the E score comes from PSI-BLAST. Both of these databases search for proteins similar to the protein of interest. Hen egg white lysozyme is very close to human lysozyme based on these scores and an overlay of the tertiary/quaternary structures. Both have four alpha helices and a beta pleated sheet in the same locations and orientations, but the hen lysozyme has a triple stranded, antiparallel beta pleated sheet where the human lysozyme is only double stranded. The primary structure differs by 51 of 130 residues and an insertion of a glycine (the human lysozyme contains the glycine, the hen enzyme does not). Of those 51 only 27 have a change in charge or a large change in size of the amino acid residue. As stated before, the HLH domain is highly conserved across all types of lysozyme. Despite the differences, both human and hen lysozyme have the same function.