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.     

Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin

The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH-ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH-cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin-NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.     

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.     

Nuclear magnetic resonance and ultraviolet difference spectral studies of the binding properties of turkey egg white lysozyme. Consequences of the replacement of Asp 101 by glycine

The nuclear magnetic resonance spectrum of the ¹⁹F nuclei in N-trifluoroacetylated chitotriose was studied in the presence of turkey lysozyme. In contrast to results previously obtained with hen lysozyme, the ¹⁹F nmr spectrum of the complex did not show any striking pH dependence. It was, in fact, very similar at all pH's to the spectrum of the trisaccharide complexed with hen lysozyme at low pH, where Asp 101 is protonated. The replacement of Asp 101 in turkey lysozyme by a glycine is thought to account for this difference and the results allow unequivocal assignment of a value of 4.2 to the pK_a of Asp 101 in hen lysozyme. The dissociation constant of the chitotriose-turkey lysozyme complex was measured at various pH's using uv difference methods and compared with that previously reported for the hen lysozyme-chitotriose complex. Again, the results could be attributed to the loss in binding energy due to the absence of Asp 101. In contrast to chitotriose, the binding of chitobiose and methyl-2-acetamido-2-deoxy-β-d-glucopyranoside as studied by both uv difference and nmr methods is the same within experimental error for turkey and hen lysozyme. The results obtained for binding of chitobiose suggest that Asp 101 does not contribute as much to the binding energy of the disaccharide as was previously thought. Finally, the specific activities of both of these lysozymes against Micrococcus lysodeikticus were found to be identical.     

Protein modification in nonaqueous media. Preparation and properties of matrix-supported lysozyme

Hen's egg white lysozyme (EC 3.2.1.17) has been covalently attached to a polystyrene matrix via interaction of protein nucleophiles with an aromatic imidazolide function under anhydrous conditions. The polymer-enzyme complex is prepared in a way which allows nonaqueous solubilization of the complex. The activity of the bound enzyme compares favorably with the activity of the native protein. The pH optima for the matrix-supported protein are shifted toward the basic side. The effect of substrate concentration on rate has been determined. (A preliminary report of this work has been published: G. J. Bartling, H. D. Brown, S. K. Chattopadhyay, Nature 243 , 342–344 (1973).)     

Preparation and properties of an immobilized derivative of penicillinase.

Penicillinase (beta-lactamase I, EC 3.5.2.6) secreted by Bacillus cereus, strain 569/H, was covalently attached to aminoethyl cellulose via glutaraldehyde. The immobilized derivative shows increased thermostability and decreased susceptibility to conformational changes induced by certain substrates of penicillinase. The decline in the rate of hydrolysis of such substrates was consequently suppressed by immobilization. A marked increase in Km was observed with all substrates except for the unsubstituted 6-aminopenicillanic acid. The altered properties of the new derivative are attributed to the constraint imposed by immobilization on the conformational flexibility of the enzyme molecule. Thus, apart from obvious technological interest, immobilized penicillinase provides a useful model for the study of the role of flexibility in the function of an enzyme.     

Evidence for two adenine derivative receptors in rat ileum which are not involved in the nonadrenergic, noncholinergic response.

The receptors mediating inhibition of the rat ileum by adenosine and adenine nucleotides were studied. ATP and ADP were more potent than AMP or adeonsine. Theophylline antagonized the effects of adenosine and AMP but not those of ATP or ADP. Preparations desensitized to ATP or ADP were still inhibited by adenosine and vice versa. The nonadrenergic, noncholinergic inhibition produced by field stimulation or nicotine was not attenuated by the presence of theophylline or desensitization to ATP. These data indicate that more than one adenine derivative receptor is present in rat ileum and that ATP and adenosine are unlikely candidates for the unknown transmitter.     

Stopped-flow chemical modification with N-bromosuccinimide: a good probe for changes in the microenvironment of the Trp 62 residue of chicken egg white lysozyme

The stopped-flow chemical modification with N-bromosuccinimide (NBS) of Trp 62 of hen (chicken) egg white lysozyme (EC 3.2.1.17) was found to depend greatly on pH: it was not observed at pH's above 7, but it was observed at pH's lower than 6. In addition, at pH's between 6 and 7 the NBS modification showed a delta epsilon pH profile similar to a "titration curve," giving a pK (congruent to 6.5) nearly equal to the pK (congruent to 6.2) of a catalytic residue, Glu 35. The stopped-flow chemical (NBS) modification of N-acetyl-L-tryptophan ethyl ester, a model compound of Trp 62, does not depend on pH at the pH's examined, approximately 3.5-8.5. These experimental results suggest that a change in the state of Trp 62 at Subsite C is induced by protonation-deprotonation of an ionizable residue, which could be Glu 35 (catalytic site), indicating that stopped-flow NBS modification is a good probe for detection of changes in the micorenvironment around the tryptophan residue(s) of enzymes.     

Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase.

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.     

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin.

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups.

Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in lysozyme.     

Purification and catalytic properties of a cross-linked complex between adrenodoxin reductase and adrenodoxin

A stable covalent complex was prepared by cross-linking adrenodoxin reductase with adrenodoxin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was purified extensively until free components were removed completely. The major component of the complex had a molecular weight of 63 kDa, which corresponds to a 1:1 stoichiometric complex between adrenodoxin reductase and adrenodoxin. NADPH-cytochrome c reduction activity of the covalent complex was comparable to that of an equimolar mixture of adrenodoxin reductase and adrenodoxin (native complex), and the NADPH-ferricyanide reduction activity of the complex was equal to that of the native one. In contrast to the native complex, the covalent complex produced much less superoxide upon NADPH-oxidation, and the covalent complex was found to be more stable than the native complex, suggesting that the complex state is more favorable for catalysis. From these results, we conclude that the adrenodoxin molecule does not need to dissociate from the complex during electron transfer from NADPH to cytochrome c.     

A two-dimensional NMR study of exchange behavior of amide hydrogens in a lysozyme derivative with an extra cross-link between Glu35 and Trp 108—quenching of cooperative fluctuations and effects on the protein stability

Two dimensional nmr spectra [correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA), nuclear Overhauser effect spectroscopy (NOESY)] have been observed for cross-linked lysozyme, a chemically modified lysozyme derivative with an extra ester cross-link between residues E35 and W108. Eight shifted cross-peaks were found in the fingerprint region of COSY spectra. By searching COSY, HOHAHA and NOESY spectra, they have been assigned to A32, E35, S36, I58, A107, W108, V109, and A110. The NOE connectivities (d_NN and d_αN) found for the cross-linked lysozyme are quite similar to those for the intact lysozyme. Exchange behavior of amide hydrogens has been studied for both intact and cross-linked lysozymes by observing the fingerprint region of COSY spectra. Hydrogen exchange reactions were carried out at pH 7.0 and at several temperatures. There exist 41 amide hydrogens whose exchange reactions are detectable under this experimetal conditon. Not only exchange rates but also their activation enthalpies were determined for individual amide hydrogens. They are classified into two groups, which are called categories III and IV. Category III hydrogens are distributed in relatively flexible peripheral parts of protein, and category IV hydrogens are deeply buried in the core region of protein. Category III hydrogens are exchanged through localized unfolding around their sites with a low activation enthalpy ranging from 10 to 25 kcal/mol. The formation of an extra cross-link affects neither the exchange rate nor the activation enthalpy of category III hydrogens. However, amide hydrogens of residues 34–39 in the vicinity of the hinge are exceptions. They are easily exchanged in the intact lysozyme but their exchange rates are drastically retarded by cross-linking. In the intact lysozyme, structural fluctuations mediating the exchange of category IV hydrogens are highly cooperative with a large activation enthalpy. These large-scale structural fluctuations are the global unfolding of the overall structure and also concerted motions within a domain. Especially near 38°C, it was found that the dominant fluctuation occurring in the α-domain is different from that in the β-domain. However, these concerted motions are strongly quenched by the formation of the cross-link because of the cooperativity of such a large-scale fluctuation. The stabilization of a localized area of protein by cross-linking results in the great suppression of large-scale and concerted motions. The exchange rates of category IV hydrogens are extremely retarded in the cross-linked lysozyme, so that they are exchanged through the so-called penetration mechanism characterized by a low activation enthalpy. These expeimental results are discussed with regard to the contribution of cross-linking to the stabilization of the folded structure of protein. © 1997 John Wiley & Sons, Inc.     

Preparation and some properties of a derivative of wheat germ agglutinin with altered biological activity

An inactive derivative of wheat germ agglutinin, which is a strong activator of blood platelets, was prepared by selective chemical modification of the lectin with cyanogen bromide at acid pH. The derivative was then used as a probe to learn about the initial events in platelet stimulation by physiological agents. Amino acid analysis of the modified lectin confirmed specific cleavage of a methionine residue. Gel filtration studies indicated a molecular weight for the lectin derivative similar to the unmodified lectin. In gel electrophoresis in the presence of sodium dodecyl sulfate, reduced samples of the derivative showed two bands and the main component migrated slightly faster than the native lectin. The derivative retained the capacity to precipitate an antibody to the lectin although at least one of the antigenic sites was lost due to chemical modification. The derivative did not compete with the unmodified lectin for binding to platelets. Unlike the parent lectin, the derivative did not aggregate platelets even at a ten fold higher concentration. Under similar conditions, there were about 1.0 X 10(5) binding sites/platelet for the lectin derivative with an apparent dissociation constant of 1.7 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. Overnight incubation of platelets or red cells with the derivative in microtiter plates showed about 2-5% agglutinating activity for the derivative compared to the unmodified lectin. Incubation of platelets with the lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by a number of other agents was not significantly affected. This inhibitory effect of the lectin derivative on thrombin-induced platelet aggregation could be readily reversed with GlcNAc. The lectin derivative may be a useful tool to explore the structure-function relationship of cell surface components.     

Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two beta-propeller domains.

The ubiquitous use of heme in animals poses severe biological and chemical challenges. Free heme is toxic to cells and is a potential source of iron for pathogens. For protection, especially in conditions of trauma, inflammation and hemolysis, and to maintain iron homeostasis, a high-affinity binding protein, hemopexin, is required. Hemopexin binds heme with the highest affinity of any known protein, but releases it into cells via specific receptors. The crystal structure of the heme-hemopexin complex reveals a novel heme binding site, formed between two similar four-bladed beta-propeller domains and bounded by the interdomain linker. The ligand is bound to two histidine residues in a pocket dominated by aromatic and basic groups. Further stabilization is achieved by the association of the two beta-propeller domains, which form an extensive polar interface that includes a cushion of ordered water molecules. We propose mechanisms by which these structural features provide the dual function of heme binding and release.