Accessibility of tryptophan residues in immunoglobulin M molecule as an indicator of its conformational variability

The accessibility of tryptophan residues in immunoglobulin M to modification with the Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) was used as an indicator of its conformational variability. Of 14 tryptophan residues (per HL-fragment) in the native IgM, only one (presumably Trp312 in the mu-chain) was the most accessible. Irreversible acid- or temperature-induced conformational changes of IgM increased almost 2-fold the number of accessible tryptophan residues. After partial enzymatic deglycosylation of IgM (especially by an intense splitting of mannose), all tryptophan residues became inaccessible. Modification of the most accessible tryptophan residue increased 2- to 3-fold the number of tyrosine residues accessible to nitration with tetranitromethane. Using the spin label method, it was demonstrated that modification of four tryptophan residues in IgM considerably decreased the mobility of the Cmu 3 domain together with an essential drop in. the solubility of the modified IgM.     

The pH dependence of binding of alpha,alpha'-dibromo-p-xylenesulfonic acid to lysozyme.

The chemical modification of lysozyme (I) has been accomplished with alpha, alpha'-dibromo-p-xylenesulfonic acid (DBX) at five different pH values. I was alkylated by DBX at room temperature (28 degrees C) with decrease in enzyme activity. The rate of inactivation depended upon the pH at which alkylation was carried out. The highest rate was seen at alkaline pH values; the lowest at more acidic pH values. Amino acid analyses showed that-two lysines and two tryptophan residues had been modified at pH 9; two lysines, one tryptophan and one methionine had reacted at pH 8. A histidine residue was bound at pH 6.5 together with a tryptophan residue. At the lower pH values (2.7, 4.5, 6.5), alkylation occurred with a single tryptophan residue each. Fluorescence and CD data both ruled out the participation of tryptophans 62 or 108. Labeling experiments showed that two residues of DBX-35S were bound per molecule of I at both pH9 and pH8; one residue of DBX was bound per molecule of I at the other pH values. Sedimentation coefficients were characteristic of native lysozyme. The stoichiometry of binding and residue modification indicated that intra-molecular cross links were established. The pH dependence of the cross-linking provides means to measure several allowed intra-molecular distances. The results presented here are consistent with the existence of side chain motion in lysozyme.     

The beta-lactamase of Enterobacter cloacae P99. Chemical properties, N-terminal sequence and interaction with 6 beta-halogenopenicillanates.

The beta-lactamase of Enterobacter cloacae P99 consists of one polypeptide chain of Mr 39000 devoid of disulphide bridges and free thiol groups. It contains an unusually high proportion of tyrosine and tryptophan. The N-terminal sequence exhibits overlaps with the tryptic peptide obtained after labelling the active site with 6 beta-iodopenicillanate. The active-site serine residue is at position 64. The homology with the chromosomal beta-lactamase of Escherichia coli K 12 (ampC gene) is lower within the 25 residues of the N-terminal portion than around the active-site serine residue. The P99 beta-lactamase is inactivated by 6 beta-bromo- and 6 beta-iodo-penicillanate, with a second-order rate constant of 110-140M-1 X s-1 at 30 degrees C and pH 7.0, a value that is much lower than that observed with class-A beta-lactamases.     

Chemical Modification of Tryptophan Residues in Castor Bean Hemagglutinin

Modification of tryptophan residues in castor bean hemagglutinin (CBH) with N-bromosuccinimide (NBS) was investigated in detail. Tryptophan residues accessible to NBS increased with lowering pH and six tryptophan residues/mol were oxidized at pH 3.0, while two tryptophan residues/mol were oxidized at pH 5.0. From the pH-dependence curve for tryptophan oxidation, we suggest that the extent of modification of tryptophan in CBH is influenced by an ionizable group with pK_a = 3.6. The saccharide-binding activity was decreased greatly by modification of tryptophan concomitantly with a loss of fluorescence. A loss of the saccharide-binding activity was found to be principally due to the modification of two tryptophan residues/mol located on the surface of the protein molecule. In the presence of raffinose, two tryptophan residues/mol remained unmodified with retention of fairly high saccharide-binding activity. The results suggest that one tryptophan residue is involved in each saccharide-binding site on each B-chain of CBH.     

Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase. Effect on structure and enzyme activity

Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits. The alpha-subunit contains a histidine residue that is phosphorylated during the reaction. The beta-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141-172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit. In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isoamylamine as the Possible Neuroactive Metabolite of l-Leucine

The states of tryptophan residues in Abrus precatorius agglutinin (APA) were analyzed by chemical modification and solvent perturbation UV-difference spectroscopy. The number of tryptophan residues available for N-bromosuccinimide (NBS) oxidation increased with lowering pH, and 20 out of the 24 tryptophans in APA were modified at pH 3.0, while 2 tryptophans were eventually oxidized at pH 5.0. Modification of tryptophan greatly decreased the binding of APA with saccharides, and only 4% of the hemagglutinating activity was retained after modification of 4 tryptophan residues/molecule. When the modification was done in the presence of lactose or galactose, 2 tryptophan residues/molecule remained unmodified with a retention of a fairly high hemagglutinating activity. The data from solvent perturbation UV-difference spectroscopy indicated that 6 tryptophans were on the surface of the APA molecule, and 4 tryptophan residues/molecule were shielded from the perturbing effect of the solvent upon binding with lactose.

Based on these results, we proposed that in the saccharide-binding site on each B-chain of APA there exists one tryptophan residue directly involved in saccharide binding, and near the binding site there is another tryptophan residue whose state is also changeable upon binding with saccharide.     

Simple Synthesis of (R)-5-Hexadecanolide

Chemical modification of tryptophan residues in abrin-a with N-bromosuccinimide (NBS) was studied with regard to saccharide-binding. The number of tryptophan residues available for NBS oxidation increased with lowering pH, and 11 out of the 13 tryptophan residues in abrin-a were eventually modified with NBS at pH 4.0, while 6 tryptophan residues were modified at pH 6.0 in the absence of specific saccharides. Modification of tryptophan residues at pH 6.0 greatly decreased the saccharide-binding ability of abrin-a, and only 2% of the hemagglutinating activity was retained after modification of 3 residues/mol. When the modification was done in the presence of lactose or galactose, 1 out of 3 residues/mol remained unmodified with a retention of a fairly high hemagglutinating activity. However, GalNAc did not show such a protective effect. NBS-oxidation led to a great loss of the fluorescence of abrin-a, and after modification of 3 tryptophan residues/mol, the fluorescence intensity at 345 nm was only 38% of that of the unmodified abrin-a. The binding of lactose to abrin-a altered the environment of the tryptophan residue at the saccharide-binding site of abrin-a, leading to a blue shift of the fluorescence spectrum. The ability to generate such fluorescence spectroscopic changes induced by lactose-binding was retained in the derivative in which 2 tryptophan residues/mol were oxidized in the presence of lactose, but not in the derivative in which 3 tryptophan residues/mol were oxidized in the absence of lactose. Importance of the tryptophan residue(s) in the saccharide-binding of abrin-a is suggested.     

Structural role of histidine residues in NAD(P)-glutamate dehydrogenase from the bovine liver

Photooxidation of bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3) in the presence of methylene blue at a low light intensity occurs in two stages. At the first stage, the duration of which depends on temperature and dye concentration, a slight activation is observed simultaneously with the oxidation of two histidine residues. At the second stage, the inactivation is concomitant with the oxidation of three histidine and one tryptophan residues. The inactivation is a first order reaction (k = 3,22 X 10(-2) min-1) and is correlated with changes in the circular dichroism spectra. These data testify to the structural role of histidine residues in the GDH molecule. The kinetic behaviour of GDH during its modification with diethylpyrocarbonate (DEP) depends on pH and the reagent concentration. Four histidine residues undergo carbethoxylation at pH 6.0 and 7.5, but the modification rate is much higher at pH 7.5. At low DEP concentrations, a remarkable activation is observed with a simultaneous modification of one histidine residue, which is independent of pH. At high DEP concentrations, a rapid inactivation takes place at pH 7.5. Treatment of the carbethoxylated inactive enzyme with hydroxylamine results in the deacylation of histidine residues without any noticeable reactivation. The data on the combined effect of DEP and pyridoxal-5'-phosphate suggest that GDH inactivation by DEP at pH 7.5 is a result of modification of an essential epsilon-NH2 group of lysine-126.     

色氨酸残基在内切葡聚糖酶分子中的作用

内切葡聚糖酶的化学修饰研究表明：色氨酸残基可能位于活性位点,与底物结合有关．荧光光谱测定指出该酶的荧光几乎都来自色氨酸残基,酶分子中色氨酸微环境对ｐＨ变化非常敏感,降低ｐＨ导致了酶分子构象发生了较大变化,配基结合使酶分子色氨酸微环境产生了改变,引发了与ｐＨ诱导不同的构象变化．     

Tryptophan residue essential for activity of Naja naja atra phospholipase A2

When Naja naja atra phospholipase A2, which contains three tryptophan residues at the 18th, 19th, and 61st positions, was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased in a convex manner with increase in the extent of oxidation of tryptophan residues. The curve shape showed that the tryptophan residue oxidized last is most responsible for the activity. The order of accessibilities of the three tryptophan residues, which was analyzed according to the method reported previously (Mohri et al. (1876) J. Biochem. 100, 883-893), was Trp-61 greater than Trp-19 greater than Trp-18. Thus, Trp-18 was evaluated to be essential for activity. Difference spectra of phospholipase A2 produced by titrating with laurylphosphorylcholine in the presence of Ca2+, which are due in large part to perturbation of the tryptophan residue(s), were retained with phospholipase A2 derivatives containing 1.2 and 2.0 mol of tryptophan residues oxidized but not with the derivative containing 3.0 mol of tryptophan residues oxidized. Such observations led us to assume that Trp-18 is involved in the specific site that interacts with phospholipid.     

Significance of the Tryptophan Residue at the Low Affinity Saccharide-binding Site of Ricin E

Chemical modification of tryptophan residues in ricin E was investigated with regard to saccharide-binding. Two out of ten tryptophan residues in ricin E were modified with N- bromosuccinimide at pH 4.5 in the absence of specific saccharide accompanied by a marked decrease in the cytoagglutinating activity. Such a loss of the cytoagglutinating activity was found to be principally due to the oxidation of one tryptophan residue per B-chain. In the presence of lactose, one tryptophan residue/mol was protected from the modification with retention of a fairly high cytoagglutinating activity. However, G a IN Ac did not show such a protective effect. The binding of lactose to ricin E altered the environment of the tryptophan residue at the low affinity binding site of ricin E, leading to a blue shift of the fluorescence spectrum and an UV-difference spectrum with a maximum at 290 nm and a trough at 300 nm. The ability to generate such spectroscopic changes induced by lactose was retained in the derivative in which one tryptophan residue/mol was oxidized in the presence of lactose, but not in the derivative in which two tryptophan residues/mol were oxidized in the absence of lactose. Based on these results, it is suggested that one of the two surface-localized tryptophan residues is responsible for saccharide binding at the low affinity binding site of ricin E, which can bind lactose but lacks the ability to bind GalNAc.     

Identification of the Tryptophan Residue Located at the Substrate-binding Site of Rye Seed Chitinase-c

Chemical modifications of rye seed chitinase-c (RSC-c) with various reagents suggested the involvements of tryptophan and glutamic/aspartic acid residues in the activity. Of these, the modification of tryptophan residues with N-bromosuccinimide (NBS) was investigated in detail.

In the NBS-oxidation at pH 4.0, two of the six tryptophan residues in RSC-c were rapidly oxidized and the chitinase activity was almost completely lost. On the other hand, in the NBS-oxidation at pH 5.9, only one tryptophan residue was oxidized and the activity was greatly reduced. Analyses of the oxidized tryptophan-containing peptides from the tryptic and chymotryptic digests of the modified RSC-c showed that two tryptophan residues oxidized at pH 4.0 are Trp72 and Trp82, and that oxidized at pH 5.9 is Trp72.

The NBS-oxidation of Trp72 at pH 5.9 was protected by a tetramer of N-acetylglucosamine (NAG₄), a very slowly reactive substrate for RSC-c, and the activity was almost fully retained. In the presence of NAG4, RSC-c exhibited an UV -difference spectrum with maxima at 284 nm and 293 nm, attributed to the red shift of the tryptophan residue, as well as a small trough around 300 nm probably due to an alteration of the environment of the tryptophan residue. From these results, it was suggested that Trp72 is exposed on the surface of the RSC-c molecule and involved in the binding to substrate.     

An essential tryptophan residue for rabbit muscle creatine kinase

The tryptophan residues in rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been modified by dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide after reversible protection of the reactive SH groups. The modification of two tryptophan residues as measured by spectrophotometric titration leads to complete loss of enzymatic activity. Control experiments show that reversible protection of the reactive SH groups as S-sulfonates followed by reduction results in nearly quantitative recovery of enzyme activity. The presence of a 410 nm absorption maximum and the decrease in fluorescence of the modified enzyme indicate the modification of tryptophan residues. At the same time, SH determinations after reduction of the modified enzyme show that the reagent has not affected the protected SH groups. Quantitative treatment of the data (Tsou, C.-L. (1962) Sci. Sin. 11, 1535 1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of substrates partially protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.     

Accessibility of tryptophan residues in immunoglobulin M as an index of its conformational changeability

Demonstrated herein is the possibility of using the accessibility of tryptophan (Trp) residues in immunoglobulin M (IgM) upon modification with Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) as an index of the conformational changeability of IgM. Of fourteen Trp's in the native IgM (per HL-region) only one appeared to be most accessible, evidently Trp312 in the mu-chain. Irreversible acidic and thermal conformational transitions in IgM increase the number of accessible Trp's approximately two-fold. Following partial enzymatic deglycosylation of IgM, deep scission of mannose in particular, all Trp's become inaccessible. Modification of the most accessible Trp increases 2-3 fold the number of tyrosine residues readily accessible upon nitration with tetranitromethane. Modification of four trp's using spin-label method data causes a sharp reduction of the mobility of the C mu 3 domain and a simultaneous decrease in the solubility of modified IgM.     

Proton-nuclear-magnetic-resonance study of the conformation of neurotoxin II from Middle-Asian cobra (Naja naja oxiana) venom.

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle-Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2-8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances. The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.     

Chemical modification of dipeptidyl peptidase iv: involvement of an essential tryptophan residue at the substrate binding site

Inactivation of pig kidney dipeptidyl peptidase IV (EC 3.4.14.5) by photosensitization in the presence of methylene blue at pH 7.5 was observed to have pseudo-first-order kinetics. During the process, until over 95% inactivation was achieved, the histidine and tryptophan residues were decreased from 14.0 to 2.7 and 12.6 to 7.1, respectively, per 94,000-Da subunit, without any detectable changes in other photosensitive amino acids. Modification of four histidine residues per subunit using diethylpyrocarbonate resulted in only 30% inactivation of the enzyme, while N-bromosuccinimide almost completely inactivated the enzyme with the modification of only one tryptophan residue per subunit, as determined by absorption spectrophotometry at 280 nm. The protective action of the substrate and inhibitors such as Ala-Pro-Ala and Pro-Pro against the modification of tryptophan residues with N-bromosuccinimide was observed both fluorometrically and by measurement of activity. On the basis of these results it is suggested that one of the tryptophan residues in the enzyme subunit is essential for the functioning of the substrate binding site of pig kidney dipeptidyl peptidase IV.     

Conformation, stability, and folding of interleukin 1 beta

Recombinant human interleukin 1 beta has been studied in solution with respect to its conformation, stability, and characteristics of unfolding and refolding. It is an all-beta-type, stable globular protein with a high cooperativity under conditions where refolding is reversible. The tryptophan residue is approximately 40% exposed to solvent, and the four tyrosines are 50% exposed. The fluorescence of the single tryptophan residue is quenched at pH 7.5 but dequenched by high salt, by titration to lower pH with a pK of 6.59, and by denaturants, resulting in an unusual biphasic change in fluorescence on unfolding. Both histidine and thiol residues have been excluded as being responsible for the pH dependence of fluorescence by site-directed mutagenesis and by chemical modification, respectively. The likely candidate is an aspartate or glutamate.     

1,2-Cyclohexanedione modification of arginine residues in egg-white riboflavin-binding protein

1. Reaction of 1,2-cyclohexanedione with arginine residues of egg white riboflavin-binding protein results in a loss of the binding activity. 2. In borate buffer pH 8.0, with 0.15 M cyclohexanedione, the inactivation proceeds with a pseudo-first-order rate constant 0.084 hr.-1. 3. At least 65% of lost riboflavin binding capacity can be recovered on 12 hr incubation in 0.5 M hydroxylamine pH 7.0. 4. All 5 arginine residues are modified, 2-3 of them seem to react much easier than others. 5. The correlation between modification of arginines and protein inactivation, as analyzed by kinetic and statistical methods, suggests that one of low-reactivity residues is "essential" for riboflavin binding. 6. In the holoprotein, one arginine residue is almost completely protected from 1,2-cyclohexanedione modification. 7. Riboflavin does not dissociate from holoprotein, even on prolongated incubation with the reagent. 8. The protected arginine residue seems to be located in the riboflavin binding pocket of protein macromolecule.     

Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies.

When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.     

Structural studies of MS2 bacteriophage virus particle disassembly by nuclear magnetic resonance relaxation measurements

In this article we studied, by nuclear magnetic resonance relaxation measurements, the disassembly of a virus particle-the MS2 bacteriophage. MS2 is one of the single-stranded RNA bacteriophages that infect Escherichia coli. At pH 4.5, the phage turns to a metastable state, as is indicated by an increase in the observed nuclear magnetic resonance signal intensity upon decreasing the pH from 7.0 to 4.5. Steady-state fluorescence and circular dichroism spectra at pH 4.5 show that the difference in conformation and secondary structure is not pronounced if compared with the phage at pH 7.0. At pH 4.5, two-dimensional (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum shows approximately 40 crosspeaks, corresponding to the most mobile residues of MS2 coat protein at pH 4.5. The (15)N linewidth is approximately 30 Hz, which is consistent with an intermediate with a rotational relaxation time of 100 ns. The average spin lattice relaxation time (T(1)) of the mobile residues was measured at different temperatures, clearly distinguishing between the dimer and the equilibrium intermediate. The results show, for the first time, the presence of intermediates in the process of dissociation of the MS2 bacteriophage.