Search

Two-component phosphorelay systems are minimally consisted of a sensory protein-histidine kinase (HK) and a response regulator (RR). HK autophosphorylates its conserved histidine residue in response to stimulus from an

When monitored by 1H NMR at various pH values, most of the C-2 proton signals from 12 His residues of the isolated beta subunit of thermophilic F1-ATPase (TF1) could be separately observed. Two of them were assigned to His-179 and His-200 which reside at the entrance of a 'conical tunnel' to reach catalytic site in the crystal structure of F1-ATPase. His-200 gave doublet, suggesting that this region is not a rigid alpha-helix in the isolated beta subunit. The binding of Mg.AMP-PNP changed the chemical shifts of His-179 and His-200 significantly. Although His-119 located at the opposite side of the conical tunnel was not affected by the nucleotide-binding, it contributed to the stability of beta subunit and the efficiency of the catalysis of the holoenzyme.

Treatment of anti-ovalbumin rabbit IgG with diethylpyrocarbonate (DEPC) at concentrations up to 100 μM led to a progressive decrease in the rates of formation of insoluble immune complexes, without affecting the final extent of immune complex formation. DEPC concentrations approximately 10-fold higher were needed to give comparable decreases in the rates of immune complex formation by F(ab′) 2 . Treatment of DEPC-treated IgG with hydroxylamine led to substantial restoration of the rates of formation of insoluble immune complexes. Carbethoxylation of two histidine residues per IgG molecule had little effect on rates of formation of insoluble immune complexes, but these rates were markedly decreased in samples of IgG with four to five histidines per molecule modified. There were parallel decreases in the protein A-binding activity and in the rates of formation of insoluble immune complexes in IgG treated with increasing concentrations of DEPC. The presence of complement protein C1q restored the rates of formation of insoluble immune complexes of DEPC-treated IgG.

The inositol monophosphatase from bovine brain is inactivated by the histidine-specific reagent diethylpyrocarbonate. Using 4 mM reagent at pH 6.5, the reaction results in the modification of 3 equivalents of histidine per polypeptide chain. The loss of activity occurs at the same rate as the slowest reacting of these residues. Site directed mutagenesis studies have been used to generate a mutated enzyme species bearing a His-217→Gln replacement and have shown that it is the modification of histidine 217 which results in the inactivation of the enzyme.

Purified ADPRT protein was inactivated by the histidine specific reagent diethylpyrocarbonate, binding to two histidine residues, or by a relatively histidine selective photoinactivation method. Inactivation with up to 1.3 mM diethylpyrocarbonate was reversible by hydroxylamine. Enzymatic inactivation coincided with the loss of binding capacity of the enzyme protein to benzamide affinity matrix but not to DNA cellulose. Labelled diethylpyrocarbonate was identified exclusively in the 56 kDa carboxyl-terminal polypeptide where 2 out of 13 histidine residues were modified by this reagent. It is proposed that histidine residues in the 56 kDa polypeptide may participate as initiator sites for polyADP-ribosylation.

BIOCHEMISTRY Two-component systems enable microbes to respond to environmental conditions. Signal transduction begins when a dimeric integral membrane protein senses an external signal, and one of the monomers phosphorylates the other on a histidine residue. Thisphosphoryl group is then transferred

Formaldehyde released during hydrogen fluoride deprotection of peptides containing a histidine residue protected by the Nπ-benzyloxymethyl (Bom) group induces cyclisation of N-terminal cysteine to thiazolidine carboxylic acid (Thz).

The Nim-2-adamantyloxycarbonyl (2-Adoc) group is suitable for the protection of the imidazole function of the histidine residue in peptide synthesis in terms of its stability to trifluoroacetic acid (TFA), tertiary amines and 1-hydroxybenzotriazole (HOBt), and its reduction of racemization rate during the coupling reaction.

The pH sensitivity of benzodiazepine binding suggests that a histidine residue may be present in, or close to the benzodiazepine binding site. This was confirmed by the selective modification of histidine residues using diethyl pyrocarbonate which was found to block both benzodiazepine and beta-carboline binding. In order to assess whether this histidine residue is located in or adjacent to the benzodiazepine and beta-carboline binding sites, experiments were performed using either benzodiazepine or beta-carboline to protect against diethyl pyrocarbonate treatment. It was found that benzodiazepine agonists, but not propyl beta-carboline protect the benzodiazepine binding sites from diethyl pyrocarbonate modification.

The 'H and 13C NMR peaks from the imidazole rings of the two histidine residues in Anabaena variabilis ferredoxin I1 (histidine-16 and histidine-93) were assigned by reference to spectra of the homologous ferredoxin from Spi- rulina maxima which contains only one histidine residue (histidine-93). The conserved histidine-93 has unusual properties: its pKL in the oxidized protein is abnormally low (5.3 in A. variabilis ferredoxin and 5.9 in S. maxima ferre

The synthesis is described of β-corticotrophin-(1–24)-tetracosapeptide ‡ labelled with tritium in the histidine residue at position 6 at a specific radioactivity of 30 Ci mmol–1 by reductive dehalogenation of a protected precursor. Evidence for the integrity of the final product is provided by amino-acid analysis, column chromatography, and bioassay, supported by chemical and enzymic analytical data on the protected precursor and the derived free peptide containing di-iodohistidine.

1. When thermolysin was treated with a 100-fold molar excess of 2,4,6-trinitrobenzene-1-sulfonate at pH 8.0 and 37 degrees for 7 h, all 12 amino groups in the enzyme were almost completely trinitrophenylated. The fully trinitrophenylated enzyme still retained more than 80% of its original activity. The amino groups are therefore not essential for activity. 2. When treated with a 100- to 1,000-fold molar excess of N-acetylimidazole at pH 6.5 and 23 degrees for 2 h, thermolysin lost about 54% of its activity with concomitant acetylation of 21 tyrosine residues out of the total of 28 residues. The reaction did not easily proceed any further. This partially inactivated enzyme regained almost full activity upon treatment with hydroxylamine. These modified tyrosine residues are therefore not directly involved in the active site. 3. Thermolysin was not inactivated by treatment with about 100- to 150-fold molar excess of 2-hydroxy-5-nitrobenzyl bromide or dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide at pH 6.0 and room temperature for 1 h both in the presence and absence of 8 M urea. Thus the three tryptophan residues in the enzyme are not accessible to these reagents. When treated with a 4.3- to 43-fold molar excess of N-bromosuccinimide over tryptophan, the enzyme was inactivated to varying extents, depending on the reaction conditions used. In this case, the tyrosine residues appeared to be the most rapidly modified, but tryptophan and histidine residues were also modified with extensive inactivation at higher concentrations of the reagent. The presence of 8 M urea retarded the inactivation.

The complete amino acid sequence of the myoglobin from Aplysia kurodai, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 144 amino acid residues, is acetylated at the amino terminus and contains one histidine residue at position 95. It is also of interest that the myoglobin differs in amino acid sequence from that of Aplysia limacina, a Mediterranean species, by 21 replacements and one deletion.

Electrostatic calculations for human rhinovirus 14 indicate that histidine-base residue pairs in the region of a β-strand interaction between pentamers may be involved in a pH-induced process that leads to the release of viral RNA. Other picornavirus sequences are examined for these residue pairs, a subset of which is present in enteroviruses. Foot and mouth disease virus possesses one of the residue pairs, and cardioviruses, which undergo a separate pH and halide ion-induced capsid dissociation, possess none.

alpha- and beta-melanocyte-stimulating hormones (alpha-MSH and beta-MSH) have been isolated from Xenopus laevis neurointermediate pituitary and microsequenced. Intracellular alpha-MSH is not N-acetylated after proteolytic processing of pro-opiomelanocortin in contrast to mammalian alpha-MSHs. There is a high preservation of the melanotropic amino acid sequence common to all MSHs although in Xenopus beta-MSH a histidine residue replaces the glutamic acid residue found in position 8 of mammalian beta-MSHs.

Analogues of luliberin (luteinising hormone-releasing hormone) were synthesised where the glycine residue in position 10 was replaced by either azaglycine (–NH–NH–CO–) or aza-alanine (–NH–NMe–CO–), the histidine residue in position 2 was either omitted, or replaced by D-phenylalanine or D-tryptophan, and the glycine residue in position 6 was substituted either by D-phenylalanine or D-tryptophan. These compounds were evaluated for their ability to prevent ovulation induced by luliberin in androgen-sterilised constant-oestrus rats. Compounds with the azaglycine residue in position 10 and other modifications in positions 2 and 6 showed good antagonist activity, whereas aza-alanine replacement in position 10 together with modifications in position 2 resulted in inactive compounds. The most potent analogue, [2-D-Phe-6-D-Phe-10-Azgly]-luliberin, completely inhibited ovulation induced by luliberin (0.5 µg/rat) at a dose of 15 µg/rat.

A. Introduction . . 110 (i) Properties of blue-c~pper p&t&s _ 1 1 1 1 1 :* 1 1 1 1 1 1 1 _ 110 (ii) Chemical and physical studies _ _ . _ _ . . _ _ . . . _ . . . 110 (iii) NMR studies . . . . . _ . . . . . . . . . . . . . . . 111 B. Plastocyanins . . . . . . . . _ . . . . . . . . . . . . 113 (i) Proton NMR spectroscopy . . . . . . . . . . . . . . . . . 113 (ii) Carbon-13 NMR spectroscopy . . . . . . . _ . . . _ . . . . 116 (iii) Histidine residue pH-titration curves . . (iv) Conformationai differences between oxidized andreduced~pla&cyax&s f 119 : 120 C.Azurins. . . . 122 (i) Proton NMR~pe&roscbpy : : : : : : : : 1 1 : : : : : : . 122 (ii) Carbon-13 NMR spectroscopy . . . . . . . . . . . . . . . . . 127 (iii) Distances of protein groups from the copper . . . . . . _ . . . . 131 (iv) Conformationai heterogeneity . . . . . . . . . . . . . . . . 132 D. Discussion . . . . . . . . . . . . . . . _ . . . . . . . . 133 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . 137 References . . . . . . . . . . . _ . . . _ _ _ _ . . . _ . 137

The binding of labeled batrachotoxinin-A benzoate (BTX-B) to voltage-sensitive sodium channels in broken membrane preparations of mouse cerebral cortex has been measured as a function of the pH. Specific binding is negligible at pH

The catalytic hydrolysis of enantiomeric substrates is examined using optically active dipeptide derivatives containing histidine residue in the presence of CTABr micelles. A very high stereoselectivity, kc(L)/kc(D), of 12.2 is observed in the reaction with the enantiomers of p-nitrophenyl N-methoxycarbonylphenylalanate (MocPheONp) and N-(benzyloxycarbonyl-L-leucyl)-L-histidine (ZLeuHis).

Chemical modification with diethylpyrocarbonate (ethoxyformic anhydride) was examined to demonstrate the existence of an essential histidine residue at the NADPH-binding site of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas desmolytica. Among some ligands, NADPH was noticeable in protecting the enzyme from the modification-caused inactivation. Although several amino acid residues were modified during the inactivation process, inhibition of the enzyme could be correlated with modification of a single histidine residue which was masked by addition of NADPH. The pK of the essential histidine residue was estimated to be 6.5-6.7. The Kd (Km) for NADPH of the inactivated enzyme was shown to have been increased greatly, although the Kd for substrate (p-hydroxybenzoate) was not changed.

Ethoxyformic anhydride and photooxidation were used to study the function of histidine residues in neutral proteinase from Streptomyces naraensis. Ethoxyformic anhydride completely inactivated the enzyme. However, NH2OH reactivated the ethoxyformic derivative to 100% of the value of the enzyme treated with only 40 mm NH2OH. Since NH2OH specifically deacylates N-ethoxyformylimidazole, it was shown that at least some of the histidine residues were essential for the activity. In addition, photooxidation experiments in the presence of 0.001% rose bengal confirmed that only histidine residues of the enzyme were destroyed under the designated experimental conditions. About two histidine residues per 37,000 g of protein were destroyed during the photooxidation experiments. In the presence of the competitive inhibitor carbobenzoxy-l-phenylalanine,* only one histidine residue was destroyed, which inhibitor, that one histidine residue per 37,000 g of protein was essential for the enzyme activity.

Unlike mammalian oxymyoglobins, Aplysia MbO2 is extremely susceptible to autoxidation, and its pH dependence is also unusual. Kinetic formulation has revealed that two kinds of dissociable group with pK1 = 4.3 and pK2 = 6.1, respectively, at 25 degrees C are involved in the stability property of Aplysia MbO2. In order to characterize thermodynamically these dissociation processes involved, the effect of temperature on K1 and K2 was studied by analyzing the pH dependence for the autoxidation rate of Aplysia MbO2 in 0.1 M buffer over the pH range of 4-11, and at 15, 25 and 35 degrees C. The resulting thermodynamic parameters for each group were both those to be expected for the ionization of a carboxyl group; the delta H degrees value being numerically much less than 1 kcal.mol-1, or zero in practice, but being associated with a large negative value of delta S degrees of the order of -20 cal.mol-1.K-1. Taking into account the fact that Aplysia myoglobin contains only a single histidine residue corresponding to the heme-binding proximal one, we can unequivocally conclude that the two kinds of the dissociable group involved in the unusual stability of Aplysia MbO2 must both be carboxyl groups, the protonation of these groups being responsible for an increase in its autoxidation rate in the acidic pH range.

Unlike mammalian myoglobins, Aplysia myoglobin contains a single histidine residue that most likely corresponds to the heme-binding proximal one, its oxygenated form is extremely unstable and its CD magnitude is about two-thirds that of sperm whale myoglobin. A structural prediction also indicates that Aplysia myoglobin has an unusual heme environment distinctly altered from that of the mammalian myoglobins.

1. The Zn(II)-requiring beta-lactamase from Bacillus cereus 569/H/9, which has two zinc-binding sites, was examined by 270 MHz 1H n.m.r. spectroscopy. Resonances were assigned to five histidine residues. 2. Resonances attributed to three of the histidine residues in the apoenzyme shift on the addition of one equivalent of Zn(II). 3. Although these three histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH range 6.0–9.0 in the mono-zinc enzyme. 4. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. 5. It is proposed that these three histidine residues act as zinc ligands at the tighter zinc-binding site. 6. Resonances attributed to a fourth histidine residue shift on addition of further zinc to the mono-zinc enzyme. It is proposed that this histidine residue acts as a Zn(II) ligand at the second zinc-binding site.

The principal reaction between 14C-labelled methyl bromide and the nitrogen-containing groups of wheat protein has been shown, by combined radioactive tracer-chromatographic techniques, to be with the histidine residue. Three methylated histidines are present in the hydriodic acid hydrolysate of the protein that has been exposed to methyl bromide, and these have been identified as I-N-methylhistidine, 3-N-methylhistidine and I:3-N-dimethylhistidine. The amount of reaction occurring under typical fumigation conditions is so small that the loss of the semi-essential amino-acid, histidine, is negligible.

Stress relaxation, carried out in 85 : 15 methyl alcohol–water mixture at 60°C., of gelatin films crosslinked with oxystarch or difluorodinitrobenzene may be represented by the sum of three exponential rates of stress decay. Fresh formaldehyde-crosslinked films relax rapidly and only require two such terms. The second term is shown to be due to the breaking of crosslinks. All excepting 10% of the broken crosslinks do not re-form in unstressed positions as the formaldehyde is easily able to diffuse out of the layer. This 10% is suggested as being due to crosslinks involving a histidine residue. A large percentage of the broken oxystarch crosslinks do re-form because the oxystarch is not easily able to diffuse out of the layer.

Summary 1. As part of an extensive search for abnormal hemoglobin variants in the population of the province of Manitoba, 4100 blood samples have been examined that were obtained from persons attending Deer Lodge Hospital in Winnipeg. 2. One of these contained a mixture of the normal hemoglobins A1 and A2 and an abnormal fraction with an electrophoretic mobility, at alkaline pH, approximately intermediate between that of A1 and A2. 3. Purification and characterization of the variant fraction showed that it contained an arginine in place of a histidine residue in the position next to the N-terminal residue of the beta polypeptide chain. This new variant has been designated hemoglobin Deer Lodge.

Twenty peptides, including the haemopeptide, were isolated from a limited chymotryptic digest of cytochrome c from the skeletal muscle of the emu by chromatography on Whatman phosphocellulose P-70. These peptides were purified by electrophoresis or chromatography on paper and their sequences were determined. The complete sequence of the protein was deduced by alignment of these peptides with the known sequences of several other cytochromes c. Emu cytochrome c differs from the chicken protein in 2 positions. The most interesting feature of the sequence is the substitution of asparagine for the histidine residue found in position 33 of most other cytochromes c.

The abnormal structure of hemoglobin Mkankakee has been determined by protein sequence studies to be different from that of hemoglobin A by the substitution of a tyrosine residue for the histidine residue at position 87 of the α chain. The abnormal structure of hemoglobin Mkankakee found in a Caucasian family appears to be identical with the structure of hemoglobin Miwate found in some Japanese families.

Human caeruloplasmin showed a rapid loss of p-phenylene-diamine oxidase activity when subjected to photooxidation in the presence of methylene blue. Major conformational changes did not occur. The kinetic characteristics of the loss of phenylene-diamine oxidase activity were comparable to those of the destruction of histidine residues, and the relative extent of the two processes indicated a one-to-one correspondence between them. Copper was lost at a slower rate than that of destruction of histidine residues. It is suggested that the site responsible for the oxidase activity of caeruloplasmin may contain an essential histidine residue and that, although histidine residues may also be involved in copper binding, they are not exclusive copper ligands.

The RrF 1 -ATPase from the photosynthetic bacterium Rhodospirillum rubrum was rapidly and completely inactivated by diethyl pyrocarbonate (DEPC) at pH 6.0 and 22°C. When applied in 1 step 5 mM DEPC were required for > 90% inactivation and this DEPC-modified enzyme showed an increase in absorption at 242 nm as well as a decrease in absorption at 280 nm, suggesting modification of both histidine and tyrosine residues. Complete inactivation of the RrF 1 -ATPase could be obtained with only 250 μM DEPC when applied in 5 separate steps of 50 μM each. The only absorption change observed under these conditions was an increase at 242 nm indicating that the inactivation can be correlated with modification of histidine residues. Complete inactivation requires the modification of 2–3 histidine residues per molecule of RrF 1 .

The hyperfine couplings of the remote nitrogens of histidine ligands are determined for the first time by an X-band ESEEM spectroscopy study of 15N-substituted superoxide dismutase (SOD). They show a significant difference between two groups of ligands with different orientation relative to the metal ion. The ESEEM spectra of 15N SOD with cyanide as an inhibitor containing 14N and 15N are also discussed. They allow some conclusions to be drawn about structural changes upon inhibitor binding and indicate the necessity of further multifrequency investigations.