Active human lysozyme

C.-G. Golander, V. Hlady, K. Caldwell, and J. D. Andrade, Absorption of human lysozyme and adsorbate enzyme activity as quantified by means of total internal reflection fluorescence, 125I labeling and ESCA, Colloids Surf. 50, 113-130 (1990). 

These results suggest that the crystallographic determination of the structure of a productive enzyme-substrate complex is feasible for lysozyme and oligosaccharide substrates. They also provide the information of pH, temperature, and solvent effects on activity which are necessary to choose the best conditions for crystal structure work. The system of choice for human lysozyme is mixed aqueous-organic solvents at -25°C, pH 4.7. Data gathered on the dielectric constant, viscosity, and pH behavior of mixed solvents (Douzou, 1974) enable these conditions to be achieved with precision. 

A preliminary description of the sequence of the amino acids in human lysozyme has been published, and it may be compared with the sequence in hen egg-white lysozyme.116 The human enzyme, which is 2.7 to 3.0 times as active as the hen s enzyme, was obtained... 

The active site is in a cleft between a large domain with a nonpolar core and a smaller (3-sheet domain that contains many hydrogen-bonded polar side chains (Figs. 12-3,12-4). Human lysozyme has a similar structure and properties.57-59 The T4 lysozyme has an additional C-terminal domain whose function may be to bind the crosslinking peptide of the E. coli peptidoglycan. Goose lysozyme is similar in part to both hen lysozyme and T4 lysozyme. All three enzymes, as well as that of our own tears, may have evolved from a common ancestral protein.60 On the other hand, Streptomyces erythaeus has developed its own lysozyme with a completely different structure.61 An extensive series of T4 lysozyme mutants have been studied in efforts to understand protein folding and stability.61-63... 

FIGURE 13.13. Diagram of the human lysozyme molecule, with residues having displacements greater than 0.2 M outlined by parallel lines. The active site cleft runs almost vertically down the center of the drawing. (Courtesy D. C. Phillips)... 

Blanch, E.W., Morozova-Roche, L.A., Cochran, D.A., Doig, A.J., Hecht, L., and Barron, L.D. Is polyproline II helix the killer conformation A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme, /. Mol. Biol., 301, 553, 2000. 

Pathological serum and urine samples with a high activity should be diluted. The absorbance decrease per minute caused by the sample is measured, and the activity can be read directly from the curve. It should be noted that this activity is expressed in hen egg white lysozyme (HEWL) equivalents instead of human lysozyme, which is about four times as active. However, the use of the HEWL standard in clinical analysis is common practice as long as the activity of nonpathological samples is expressed in the same way for comparison. 

The shortest peptide that has been shown to interact with the HR-N trimer is HL9 (39), a nine-residue peptide derived from human lysozyme that potently inhibits HIV-1 syncytia formation in cell culture with an EC50 of 50 nM [144,145]. HL9 (39) contains two tryptophan residues separated by two amino acids that complement the hydrophobic pocket formed by the HR-N N17 fragment [142]. However, HL9 (39) is not completely specific for HIV-1, since it also exhibits antimicrobial activity [146]. 

Lee-Huang S, Maiorov V, Huang PL et al (2005) Structural and functional modeling of human lysozyme reveals a unique nonapeptide, HL9, with anti-HIV activity. Biochemistry 44 4648-4655... 

Fig. 8.2 Comparison of the promoter activities from the promoters of different storage protein genes. Seven promoters were tested using a transgenic approach, and human lysozyme was used as reporter gene. Cff, Ct3, Club-1 and Club-2 are...

Nonpolar residues formed a hydrophobic patch in the middle of the back side (opposite the active site cleft) of both proteins as can be seen in figure 4 e f. The hydrophobic patch on the human lysozyme appeared to be slightly larger than that on the hen lysozyme. 

Figure 4. (A) Front view (looking into the active site) and (B) back view of human lysozyme colored according to Feldmann s functional color scheme. Front view of (C) human lysozymes colored according to Eisenberg s atomic solvation parameters. Tryptophan residues are additionally colored red. Continued on next page.

Data taken form Craik et al. (1987) for rat trypsin Wilks et al. (1990) for B. stereothermophilus lactate dehydrogenases Muraki et al. (1992) for human lysozyme in which the active site residues refer to E35, D53, W64, D102 and W109 and AGr = -RTIn[(koat/Kra)niutant/ (kcat/Kra)wiidl Stebbins and Kantrowitz (1992) for B. subtilis aspartate transcarbamylase Casal et al. (1987) for yeast triosephosphate isomerase Dupureur et al. (1992) for pancreatic phospholipase Mrabet et al. (1992) for Actinoplanes missouriensis xylose isomerase. 

The use of optical rotatory dispersion in quantitative analysis has been applied to the microdetermination of lysozyme. A spin-label assay for lysozyme which is based on the enzymic hydrolysis of spin-labelled peptidoglycan has been described. Hydrolysis of this polymer by lysozyme results in sharpening of the e.s.r. spectrum. According to this method, human lysozyme is 3.5 times as active as hen egg-white lysozyme. The assay is suitable for measuring lysozyme levels in biological fluids. 

Lysozymes derived from sources other than bird egg-whites were also tested for their susceptibility to inactivation by the epoxypropyl /8-gly-cosides. Among these, human leukemic urine lysozyme was inactivated by (GlcNAc)3-Ep at pH 5.5, whereas the lysozymes from papaya latex and the bacteriophage T4 were not inactivated at all. Similar results were also obtained at pH 4.6, at which the papaya lysozyme acts on chitin most efficiently. These results show that the active sites of human and the hen lysozymes are similar, whereas the active sites of the T4 phage and the papaya lysozymes differ from them. Indeed, the hen and the human lysozymes are similar in their amino acid sequences" and three-dimensional structures, whereas the papaya and the T4 phage lysozymes differ from the hen egg-w hite lysozyme in their amino acid composition, molecular weight, and substrate specificity."" ... 

Some modified human-lysozymes are designed and obtained by recombinant DNA technology. Some of them are crystalline proteins. The tertiary structures and their active sites have been investigated by means of X-ray analysis and molecular dynamics by computer-graphics as is shown in Figurer 5. 

Among the specific enzymes whose activity has been reported to be decreased after in vitro ozone exposure are papain, glyceraldehyde-3-phosphate dehydrogenase, lysozyme, ribonuclease, and acetylcholinesterase. The latter enzyme appears to be particulady susceptible to free-radical and oxidative states. A loss in acetylcholinesterase activity has been reported in the red cells of humans and mice that inhaled ozone. However, there are only minimal amounts of this enzyme in lupg tissue, and, although it has been suggested that acetylcholinesterase is important in bronchial tract ciliary activity, there is no direct evidence to support this conjecture. 

The pH optima of human milk lysozyme (HML), bovine milk lysozyme (BML) and egg-white lysozyme (EWL) are 7.9, 6.35 and 6.2, respectively. BML has a molecular weight of 18kDa compared with 15kDa for HML and EWL. The amino acid composition of BML is reported to be considerably different from that of HML or EWL. All lysozymes are relatively stable to heat at acid pH values (3-4) but are relatively labile at pH greater than 7. Low concentrations of reducing agents increase the activity of BML and HML by about 330%. 

As discussed in section 8.2.5, lysozyme has been isolated from the milk of a number of species human and equine milks are especially rich sources. In view of its antibacterial activity, the large difference in the lysozyme content of human and bovine milks may have significance in infant nutrition. It is claimed that supplementation of baby food formulae based on cows milk with egg-white lysozyme gives beneficial results, especially with premature babies, but views on this are not unanimous. 

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