Activity of crystalline turkey egg white lysozyme.

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.     

Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase.

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.     

Recovery of lysozyme from hen egg white by selective magnetic cake filtration

A new concept for the improvement of the downstream processing and purification is the so‐called magnetic separation. By using surface functionalized magnetic substrate particles, which selectively adsorb the target product, it can be directly separated out of a crude bioprocess stream. These methods are already used for analytical purposes, where only small amounts of functionalized particles are necessary. To apply the same concept at a larger scale, effective and economical procedures have to be provided. First, suitable process equipment has to be developed. Second, the magnetic particles have to be manufactured with a stable surface functionalization and long‐term stability for their reuse. Up to now mainly high‐gradient magnetic separation filter devices are applied for selective magnetic separation. They consist of a magnetic matrix in which the magnetic particles are trapped. In this work, a new magnetic filter is introduced that overcomes the capacity limitations of the current high‐gradient magnetic separation technology. The principle is demonstrated by selective recovery of lysozyme from hen egg white. Prior to the separation experiments magnetic beads with a strong acid cation‐exchange surface functionalization are synthesized. The separation procedure is implemented in only one unit operation. With the implementation of the displacement elution sequence lysozyme can be separated out of a hen egg white solution with a purification factor of PF=36 and a purity P=0.83.     

Nuclear magnetic resonance and circular dichroism studies of a triple-helical peptide with a glycine substitution.

The triple-helical conformation has the stringent amino acid sequence constraint that every third residue must be a glycine, (X-Y-Gly)n. We use nuclear magnetic resonance and circular dichroism to quantify the consequences of a substitution in the glycine position of a triple-helical peptide, and to enhance our understanding of interactions in this basic structural motif. A 30-residue peptide with a Gly----Ala change forms a stable trimer at a folding rate somewhat less than that of the unsubstituted peptide, and the substitution results in a marked decrease in thermal stability and a conformational perturbation of about 30% of the triple-helical structure. Two models were generated for this peptide, one with the alanine residues packed inside the triple helix and one with a looping out of the chain at the substitution site. Studies on the Gly----Ala peptide are useful in understanding connective tissue diseases which result from the substitution of one glycine residue in the triple-helix of fibrillar collagens.     

1H-NMR study on the chitotrisaccharide binding to hen egg white lysozyme.

Interaction between hen egg white lysozyme and chitotrisaccharide was investigated by 1H-NMR spectroscopy using partially acetylated chitotrisaccharides and chemically modified lysozyme. Monoacetyl (GlcN-GlcN-GlcNAc), diacetyl (GlcN-GlcNAc-GlcNAc), or triacetyl chitotrisaccharide [(GlcNAc)3] was added to the lysozyme solution, and the changes in the 1H-NMR signals of the lysozyme were analyzed. Although many of the resonances were affected by addition of the saccharide, the most remarkable effect was seen on the signal of Trp28 C5H which is in a hydrophobic box adjacent to the saccharide-binding site. The signal shifted upfield by 0.2 ppm upon (GlcNAc)3 binding, whereas the chemical shift change of the signal resulting from binding of GlcN-GlcNAc-GlcNAc or GlcN-GlcN-GlcNAc was smaller than that resulting from (GlcNAc)3 binding. When the Asp101-modified lysozyme was used instead of the native lysozyme, the chemical shift change of the Trp28 C5H signal resulting from (GlcNAc)3 binding was also smaller than that for the native lysozyme. The chemical shift change of the signal reflects the conformational change of the hydrophobic box region which should synchronize with the movement of the binding site resulting from the saccharide binding. Therefore, the conformational change resulting from the saccharide binding might be reduced when the sugar residues located at binding subsites A and B of the lysozyme are deacetylated, as well as when Asp101 interacting with the sugar residues at the same subsites is modified.     

An analysis of the Co2+-induced nuclear magnetic resonance perturbations of hen egg white lysozyme

A general methodology is presented for analyzing dipolar shifts induced by paramagnetic ions in the nuclear magnetic resonance (NMR) spectra of ligand molecules. The method is applied to the shift perturbations induced by Co2+ in the spectrum of hen egg white lysozyme. A hypothesis testing scheme is employed to evaluate statistically the relative precision with which the axially symmetric and non-axially symmetric forms of the dipolar shift equation fit the observed data. The assumption of axial symmetry for the magnetic susceptibility tensor of Co2+ is rejected at the confidence level of 99%. Since the results presented here are similar to those reached in our analysis of lanthanide-induced shifts, we suggest that the assumption of axial symmetry may, in general, not hold. Similar conclusions have been reached by other investigators in studies of paramagnetic metal binding to model systems. We have included the three Co2+ coordinates in an eight-parameter fit of the Co2+ shift data. The Co2+ position obtained from this fit is in statistical agreement with the position inferred from x-ray data. Thus, the analysis of shift data may furnish a means for determining the site of metal complexation in macromolecules whose structure has been determined by x-ray crystallography.     

Nuclear magnetic resonance studies of inorganic phosphate binding to yeast inorganic pyrophosphatase.

Yeast inorganic pyrophosphatase is a dimer of identical subunits. Previous work (Rapoport, T.A., et al. (1973) Eur. J. Biochem. 33, 341) indicated the presence of two different Mn2+ binding sites per subunit. In the present work, the binding of inorganic phosphate to the Mn2+-inorganic pyrophosphatase complex has been studied by 1H and 31P nuclear magnetic resonance. Two distinct phosphate sites have been found, having dissociation constants of 0.24 mM and 18 mM. The Mn2+-31P distance from tightly bound Mn2+ to phosphate bound in the low affinity site (6.2 A) is consistent with outer sphere binding. Binding to both phosphate sites can be simultaneously inhibited by the pyrophosphate analogue, hydroxymethanebisphosphonate, providing evidence for the physical proximity of these two sites. The weaker Mn2+ site is apparently far from both phosphate sites. From the magnitudes of the dissociation constants found for both phosphate and analogue binding and the recent work of P.D. Boyer and his co-workers (private communication) on enzyme-catalyzed phosphate-water exchange, it appears unlikely that the hydrolysis of enzyme-bound pyrophosphate is the rate-determining step in the overall enzymatic catalysis of pyrophosphate hydrolysis, at least when Mn2+ is the required divalent metal ion cofactor.     

Optical properties of lysozyme. pH and saccharide binding difference spectra.

Difference spectra associated with changes in pH and with binding of saccharides have been recorded for hen egg white (HEW) lysozyme, turkey egg white (TEW) lysozyme, and for the derivatives of the hen protein in which Tre-62 or Trp-108 had been oxidized specifically to oxindolealanine to give the Oxa-62 or Oxa-108-proteins. Identical pH difference spectra were obtained for HEW, TEW, and Oxa-62-lysozymes. Oxidation of Trp-108 is reflected in both the high and low pH (pH 7 versus 5 and pH 2 versus 5) difference spectra. The magnitude of the low pH difference spectrum is enhanced by binding of saccharide for HEW and Oxa-62-lysozymes but not for TEW lysozyme. The shapes and magnitudes of saccharide binding difference spectra are affected by oxidation of residues 62 or 108. These results can be interpreted in terms of the perturbations responsible for the lysozyme difference spectra. The pH 7 versus 5 difference spectrum results from perturbation by Glu-35 of Trp-108 and another tryptophan, probably Trp-63. Perturbation of Trp-108 and one or more other tryptophan residues by several carboxylate groups is responsible for the low pH difference spectra of the unliganded HEW and TEW lysozyme molecules. Perturbation of Trp-108 makes a principal contribution to the saccharide-binding difference spectrum. Perturbation of the Oxa-108 chromophore by ionization of Glu-35 or by saccharide binding produces absorbance changes in the 250 to 265 nm region.     

Participation of the catalytic carboxyls, Asp 52 and Glu 35, and Asp 101 in the binding of substrate analogues to hen lysozyme.

The interactions of the substrate analogues, GlcNAc, beta-methyl GlcNAc, (GlcNAc)2, and (GlcNAc)3, with turkey egg-white lysozyme [ED 3.2.1.17], in which the Asp 101 of hen lysozyme is replaced by Gly, were studied at various pH values by measuring changes in the circular dichroic (CD) band at 295 nm. Results were compared with those for hen egg-white lysozyme. The modes of binding of these substrate analogues to turkey lysozyme were very similar to those hen lysozyme except for the participation of Asp 101 in hen lysozyme. The ionization constants of the catalytic carboxyls, Glu 35 and Asp 52, in the turkey lysozyme-(GlcNAc)3 complex were determined by measuring the pH dependence of the CD band at 304 nm, which originates from Trp 108 near the catalytic carboxyls. The ionization behavior of the catalytic carboxyls of turkey lysozyme in the presence and absence of (GlcNAc)3 was essentially the same as that for hen lysozyme. The pH dependence of the binding constant of (GlcNAc)3 to hen lysozyme was compared with that to turkey lysozyme between pH 2 and 8. The pH dependence of the binding constant for (GlcNAc)3 to turkey lysozyme could be interpreted entirely in terms of perturbation of catalytic carboxyls. In the case of hen lysozyme, it was interpreted in terms of perturbation of the catalytic carboxyls and Asp 101 in the substrate-binding site. The pK values of Asp 101 in hen lysozyme and the hen lysozyme-(GLcNAc)3 complex were 4.5 and 3.4, respectively. The binding constants of (GlcNAc)3 to lysozyme molecules with different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated. The binding constant of lysozyme, in which Asp 52 and Glu 35 are deprotonated, to (GlcNAc)3 was the smallest. The other three species had similar binding constant to (GlcNAc)3.     

The stepwise binding of small molecules to proteins. Nuclear magnetic resonance and temperature jump studies of the binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme.

The binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme was studied by both nuclear magnetic resonance (NMR) and temperature-jump methods under comparable conditions. The NMR measurements on the inhibitor spectrum were carried out over a range of inhibitor concentrations including levels at which most of the inhibitor was bound to the enzyme. Data in this region were obtained by a novel difference method in conjunction with correlation spectroscopy. The results from the combination of both experimental techniques demonstrated the existence of a two-step binding mechanism and produced both values for all of the individual rate constants and also the NMR spectral data for the inhibitor in the two enzyme-inhibitor complexes. The later data characterize the environment experienced by the inhibitor at each stage in the binding process and thus provides both a three-dimensional and a dynamic picture of the interaction.     

Nuclear magnetic resonance studies of amphiphile hydration.

The nuclear magnetic resonance signal of water which remains unfrozen at ?25 °C in the presence of phosphatidylcholine has been used to determine the hydration of this amphiphile. The effects of cholesterol and sodium dodecylsulfate on both the area and linewidth of this signal indicate that these molecules cause significant changes in the structure of phosphatidylcholine vesicles in solution. Studies on other amphiphiles indicate that, whereas phosphatidylethanolamine has a hydration similar to phosphatidylcholine, species with just one hydrocarbon chain such as sodium dodecylsulfate and dodecyltrimethylammonium bromide have little, if any, hydration when assayed via the nuclear magnetic resonance experiment.     

Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme*

Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate‐type (i‐type) lyzozyme. These mutations restored the bell‐shaped pH‐dependency of the enzyme activity from the sigmoidal pH‐dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N‐acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.