Conversion of Trp 62 of hen egg-white lysozyme to Tyr by site-directed mutagenesis

An expression plasmid for hen egg-white lysozyme in Saccharomyces cerevisiae was constructed by inserting almost full-length cDNA (about 600 base pairs) encoding hen egg-white pre-lysozyme into a yeast expression vector, pAM 82. The hen lysozyme was expressed under the control of the repressible acid phosphatase promoter of pAM 82 in S. cerevisiae. About half of the expressed lysozyme was secreted in the yeast growth medium as a precise mature protein which exhibited specific activity consistent with that of authentic hen egg-white lysozyme. The replacement of Trp 62 of hen egg-white lysozyme with a tyrosine residue was performed by site-directed mutagenesis using a 19-mer oligodeoxyribonucleotide. The mutant lysozyme with Tyr 62 was found to exhibit enhanced bacteriolytic activity.     

13C-NMR study of the binding of [1-13C]galactose-labelled N-acetyllactosamine and [1-13C]galactose-enriched hen ovalbumin to soybean agglutinin

The binding of the tide compounds to soybean agglutinin was investigated using 13C-NMR spectroscopy. The equilibrium constant for the binding of N-acetyllactosamine was found to be smaller than that obtained for the binding of ovalbumin (1.1 X 10(3) vs. 7.4 X 10(3) M-1). Only two binding sites per lectin tetramer were determined for the binding of ovalbumin, which is half the number of binding sites reported for the binding of small ligands to the lectin. Steric interference between the bulky ovalbumin molecules is believed to be the reason for the observed decrease in the apparent number of binding sites on the lectin.     

Ni2+ binds to active site of hen egg-white lysozyme and quenches fluorescence of Trp62 and Trp108

We found that the maximum emission of the tryptophyl fluorescence of hen egg-white lysozyme is shifted from 337 to 323 nm and quenched to the extent of 55% with an increase in concentrations of NiCl2 from 0 to 2M in 50 mM Na acetate buffer (pH 4.7). In contrast, NaCl does not influence the fluorescence of lysozyme up to 2M. To elucidate the particular effects of Ni2+ on the tryptophyl fluorescence of lysozyme, we have measured the assembly behavior and secondary structure of lysozyme in various concentrations of NiCl2, and determined the structures of lysozyme crystals grown in 0.3, 0.5, and 1.0M NiCl2, respectively. The results of analytical centrifugation and circular dichroism experiments show that lysozyme keeps a monomer state and has an identical secondary structure, irrespective of NiCl2 concentrations. The crystal structures show that all crystals grown in different concentrations of NiCl2 have an identical main chain and side chain conformation. And one Ni2+ binding with Odelta atom of Asp52 in the active site and coordinating with five water molecules to form hexagonal coordination has been determined for each crystal structure. Based on these results, we have proposed that Ni2+ quenches the fluorescence of Trp62 and Trp108 due to the binding of Ni2+ to the active site of lysozyme.     

Enhanced bacteriolytic activity of hen egg-white lysozyme due to conversion of Trp62 to other aromatic amino acid residues

Tryptophan at the 62nd position (Trp62) of hen egg-white lysozyme is an amino acid residue whose action is essential for its enzymatic activity. Its indole ring may possibly come into direct contact with sugar residues of the substrate, and thus contribute significantly to substrate binding. For further elucidation of its role in catalytic processes, this amino acid was converted to other aromatic residues, such as Tyr, Phe, and His, by site-directed mutagenesis. All the mutations were found to enhance the bacteriolytic activity but to decrease the hydrolytic activity toward an artificial substrate, glycol chitin. Such a change in substrate preference appears remarkable considering the smaller size of the aromatic residue on the mutant enzyme at the 62nd position.     

Preparation and characterization of an active lysozyme derivative: Kyn 62-lysozyme.

A novel method for the preparation of Kyn 62-lysozyme, in which tryptophan 62 is replaced by kynurenine, is reported. Hen egg-white lysozyme was ozonized in aqueous solution to yield one N'-formylkynurenine residue and deformylated with hydrochloric acid in frozen solution at -10 degrees C. Crude Kyn 62-lysozyme was purified by affinity and Bio Rex 70 chromatography successively. Kyn 62-lysozyme retains affinity for chitin and is essentially an active enzyme with a slightly weakened but distinct catalytic activity. After this modification, the enzyme activity was changed differently depending on the kind of substrate. At the individual optimum pH's, lytic activity was largely retained (80% active), but the catalytic efficiency for hydrolyzing glycol chitin was relatively low (30% active). Lysis of M. lysodeikticus cell suspensions was optimally catalyzed by Kyn 62-lysozyme at pH 6.2 and at 0.088 ionic strength. These values are lower by 1.3 pH unit and 0.04 ionic strength, respectively, than those of intact lysozyme. The optimum pH and ionic strength for the hydrolysis of neutral substrates were scarcely affected. These results suggest the significance of electrostatic interaction in the lysis of lysozyme. Relatively limited loss of activity induced by modification of the 62nd residue, which is thought to participate directly in the binding of the substrate at subsite C, is discussed on the basis of the similarity of side chain structure in tryptophan and kynurenine.     

Binding of substrate analogs to hen egg-white lysozyme with an ester linkage between Glu 35 and Trp 108.

The interactions of the substrate analogs beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lysozyme. The binding constants of beta-methyl-GlcNAc and (GlcNAc)2 to 108 ester lysozyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lysozyme did not change with pH, while the binding constants to intact lysozyme decreased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when beta-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The participation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lysozyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.     

The simultaneous binding of lanthanide and N-acetylglucosamine inhibitors to hen egg-white lysozyme in solution by 1H and 13C nuclear magnetic resonance.

Lanthanide ions and the N-acetylglucosamine (GlcNAc) sugars are able to bind simultaneously to hen egg-white lysozyme (EC 3.2.1.17). The present study characterizes the properties of the ternary complexes with lysozyme, which involve up to seven paramagnetic lanthanides and two diamagnetic lanthanides, together with alpha GlcNAc, beta GlcNAc, alpha MeGlcNAc and beta MeGlcNAc. pH titrations and binding titrations of the GlcNAc sugars with lysozyme-La(III) complexes show that the GlcNAc sugars bind to at least two independent sites and that one of them competes with La(III) for binding to lysozyme. Given the known binding site of lanthanides at Asp-52 and Glu-35, the competitive binding site of GlcNAc is identified as subsite E. A simple analysis of the paramagnetic-lanthanide-induced shifts shows that the GlcNAc sugar binds in subsite C, in accordance with crystallographic results [Perkins, Johnson, Machin & Phillips (1979) Biochem. J. 181, 21-36]. This finding was refined by several computer analyses of the lanthanide-induced shifts of 17 proton and carbon resonances of beta MeGlcNAc. Good fits were obtained for all the signals, except for two that were affected by exchange broadening phenomena. No distinction could be made between a fit for a two-position model of Ln(III) binding with axial symmetry to lysozyme, according to the crystallographic result, or a one-position model with axial symmetry where the Ln(III) is positioned mid-way between Asp-52 and Glu-35. Although this work establishes the feasibility of lanthanide shift reagents for study of protein-ligand complexes, further work is required to establish the manner in which lanthanides bind to lysozyme in solution.     

Binding of [1-13C]galactose-labeled N-acetyllactosamine to Erythrina cristagalli agglutinin as studied by 13C-NMR

The equilibrium binding kinetics of enzymatically prepared N-acetyllactosamine to the lectin from Erythrina cristagalli have been investigated by 13C-NMR spectroscopy. Under the experimental conditions used, NMR signals in the spectrum, corresponding to both the free and bound disaccharide species, were observed for the first time. This has permitted the simultaneous determinations of the equilibrium binding constant and the number of binding sites per lectin molecule. At the relatively high lectin concentrations used (0.3-0.87 mM), the association constants determined at 31 degrees C (approximately 6 X 10(3) M-1) are typically lower then those obtained by other methods employing much lower lectin concentrations. Extrapolation of the experimentally observed values to infinite dilution gave a better fit of the data (Ka approximately 1.4 X 10(4) M-1) with the binding constant determined by other methods (K approximately 1.1 X 10(4) M-1). The sugar residence time on the lectin (approximately 0.2 s) was determined directly from the signal's line-width using total line-shape analysis. Similar NMR experiments may permit an analysis of the interaction of the lectin with glycoproteins and cells labelled with 13C-enriched galactose residues. Moreover, information on lectin-galactose interactions at the binding site may be obtained by using galactose labeled at various carbons.     

Hydrogen exchange in native and denatured states of hen egg-white lysozyme.

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Ketogenesis in the living rat followed by 13C-NMR spectroscopy. Infusion of [1,3-13C]octanoate

13C-NMR spectroscopy was used as a noninvasive approach to study the metabolism of [1,3-13C]octanoate in rat liver. Using a properly adjusted surface coil a liver selection of better than 90% was achieved in the intact animal without abdominal surgery. After infusion of [1,3-13C]octanoate via the jugular vein different patterns of metabolites were observed depending on the physiological state of the rat. In the fasted animal, the major metabolites were those of the Krebs cycle while in the diabetic animal ketogenic end products were predominant. As a fatty acid of medium chain length octanoate is imported into the inner mitochondrial space without control by the carnitine acyl transferase system. Hence, the metabolic differences observed between diabetic and fasted rats result from an intramitochondrial control mechanism. The in vivo 13C-NMR results therefore support previous biochemical in vitro studies which concluded that a major control of ketone body production occurs in the inner mitochondrial space, presumably via the redox potential of the liver. As an unexpected result, 13C-NMR provides evidence for the transitory esterification of the infused 13C-labeled octanoic acid. The corresponding 13C-NMR chemical shifts are typical for glycerides.     

13C-NMR spectral study of reductively [13C]methylated glycophorin B

Glycophorin BN was reductively [13C]methylated and the 13C chemical shift of the N-terminal [13C]dimethyl-leucine residue was monitored as a function of pH. These results were compared to the pH-dependent chemical shift studies of the N-terminal [13C]dimethylleucine residues of intact glycophorin AN and N-terminal glyco-octapeptide AN. The results indicate that the titration data for [13C]dimethylleucine of glycophorin BN more closely resembles the titration data observed for the [13C]dimethylleucine residue of the N-terminal glyco-octapeptide AN rather than for the [13C]dimethylleucine residue of intact glycophorin AN. Integration of the 13C resonances indicated that glycophorin BN contains 3-4 lysine residues.     

Biosynthetic preparation of L-[13C]- and [15N]glutamate by Brevibacterium flavum.

The biosynthesis of isotopically labeled L-glutamic acid by the microorganism Brevibacterium flavum was studied with a variety of carbon-13-enriched precursors. The purpose of this study was twofold: to develop techniques for the efficient preparation of labeled L-glutamate with a variety of useful labeling patterns which can be used for other metabolic studies, and to better understand the metabolic events leading to label scrambling in these strains. B. flavum, which is used commercially for the production of monosodium glutamate, has the capability of utilizing glucose or acetate as a sole carbon source, an important criterion from the standpoint of developing labeling strategies. Unfortunately, singly labeled glucose precursors lead to excessive isotopic dilution which reduces their usefulness. Studies with [3-13C]pyruvate indicate that this problem can in principle be overcome by using labeled three-carbon precursors; however, conditions could not be found which would lead to an acceptable yield of isotopically labeled L-glutamate. In contrast, [1-13C]- or [2-13C]acetate provides relatively inexpensive, readily available precursors for the production of selectively labeled, highly enriched L-glutamate. The preparation of L-[15N]glutamate from [15N]ammonium sulfate was carried out and is a very effective labeling strategy. Analysis of the isotopic distribution in labeled glutamate provides details about the metabolic pathways in these interesting organisms.