Role Of Histidine Residues Research Articles

Iron chelation by digests of insoluble chicken muscle protein: the role of histidine residues

The role of histidine residues in the chelation of iron at neutral pH by peptides from chicken muscle was investigated to see if it could contribute to the effect of muscle tissue on iron absorption. When ferric iron was chelated by L-histidine at pH 6, the ratio of iron chelated to loss of reactive histidine was 1:1. Chelation of iron by soluble peptides in a digest of insoluble chicken muscle protein was accompanied by a small loss of reactive histidine (4–7%) in the peptides. When peptides were modified with diethylpyrocarbonate, increasing loss of histidine led to a progressive decrease in iron chelation. However, even 89% loss of histidine only reduced iron chelation by 30%. It was concluded that histidyl residues do contribute to iron chelation and therefore could by involved in the promotion of iron absorption by muscle tissue. However, other amino acid residues are likely to be involved. © 2000 Society of Chemical Industry

The role of histidine residues in the HXGH site of CTP:phosphocholine cytidylyltransferase in CTP binding and catalysis.

The HXGH motif of CTP:phosphocholine cytidylyltransferase (CCT) is a unifying feature of the cytidylyltransferase family which has been proposed to function in binding of CTP and catalysis [Veitch, D. P. & Cornell, R. B. (1996) Biochemistry 35, 10743-10750]. Substitution of serine for Gly91 in the HXGH motif of CCT implicates this motif in CTP-binding [Park, Y. S., Gee, P., Sanker, S., Schuster, E. J., Zuiderweg, E. R. & Kent, C. (1997) J. Biol. Chem. 272, 15161]. The model for CTP binding involves hydrogen bond contacts between the histidine imidazole and the CTP phosphate oxygens. We have mutated His89 and His92 to Gly or Ala, which eliminate potential hydrogen bonds, and to Asn or Gln, which conserve these interactions. Mutation to Gly or Ala at both positions, and the H89Q mutation resulted in inactive enzymes. The Vmax of [N89]CT was 100-fold lower than that of wild-type CCT, but CTP binding was not perturbed, suggesting an involvement of His89 in transition-state stabilization. The H92N mutation reduced Vmax and increased the Kms for both substrates fivefold. The H92Q mutation had little effect on substrate binding or Vmax. These data suggest that the Gln92 NH2, and not the Asn NH2, is able to substitute for the histidine NH, and implicates the tau nitrogen of His92 in forming contacts with CTP. This work strengthens the hypothesis that the HXGH motif is involved in the binding of CTP and transition-state stabilization.

Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1.

Succinate transport by the rabbit Na+/dicarboxylate co-transporter, NaDC-1, expressed in Xenopus oocytes was inhibited by the histidyl-selective reagent diethyl pyrocarbonate (DEPC). Therefore the role of histidine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to alanine, but only mutant H106A exhibited a decrease in succinate transport. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decrease in Vmax with no change in Km. Cell surface biotinylation experiments showed that the transport activity of all four mutants at position 106 was correlated with the amount of cell surface expression, suggesting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport activity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be involved in determining protein expression or stability in the membrane.

Site-directed mutagenesis of histidine residues in the ethylene-forming enzyme from Pseudomonas syringae

The roles of histidine residues in the catalysis of the transformation of 2-oxoglutarate into ethylene via the ethylene-forming enzyme (EFE) from Pseudomonas syringae were studied using site-directed mutagenesis with substitution of glutamine for ten individual histidine residues. The mutant enzymes, which were expressed in Escherichia coli, were purified to homogeneity, and assayed in vitro for K m, k cat and thermostability. The relative k cat of two mutated EFEs, H305Q and H335Q, were 40% and 60%, respectively. However, a mutation at either His-189 or His-233 caused a total loss of activity, implying that these residues play important roles in the binding of iron. The k cat values for other mutant enzymes were 11-to 55-fold less than that for the wild-type enzyme. For six partially inactive mutated EFEs (but not for H305Q or H335Q), the first order rate constants for heat inactivation at 30°C were 11-to 24-fold higher than for the wild-type enzyme. It is noteworthy that the value of the first order rate constant for heat inactivation of H268Q was identical to that of H335Q. The substitution of H268 resulted in a drastic decrease of the k cat value (relative k cat was 1.8%). This suggests that the substitution at His-268 may cause the disruption of the active site of the EFE. Heat inactivation studies with the puridied mutant enzymes revealed that some mutant enzymes, such as H168Q and H116Q, were more thermolabile than the wild-type enzyme.

The role of conserved histidine residues in the pyridine nucleotide transhydrogenase of Escherichia coli.

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+, coupled to translocation of protons across the cytoplasmic membrane. The role of histidine residues in catalysis was investigated by chemical modification with diethylpyrocarbonate and by site-directed mutagenesis. Diethylpyrocarbonate inhibited both hydride ion transfer and coupled proton translocation. Histidine residues were modified as shown spectroscopically and by the ability of hydroxylamine to cause reversal of inhibition. Complete inhibition of hydride ion transfer occurred following modification of 10 residues/enzyme molecule. Site-directed mutagenesis of single conserved histidine residues or the presence of substrates did not provide resistance to inhibition by diethylpyrocarbonate. It is concluded that diethylpyrocarbonate inhibition was a consequence of the structural changes brought about by modification of many histidine residues. With the exception of beta-subunit residue His91 (beta His91), in which mutation can result in specific loss of proton translocation activity [Glavas, N. A., Hou, C. & Bragg, P. D. (1995) Biochemistry 34, 7694-7702], site-directed mutation of the remaining conserved residues alpha His450, beta His161, beta His345 and beta His354 did not demonstrate a direct role for these residues in catalysis. Mutation of beta His161 had relatively little effect on the properties of the enzyme. By contrast, mutation of alpha His450, beta His345 and beta His354 caused major loss of enzyme activities which was probably due to alterations in the structure of the enzyme. These alterations were reflected in changes in the K(m) values for transhydrogenation.

Modification of tomato and Aspergillus niger pectinesterases with diethyl pyrocarbonate.

The role of histidine residues in pectinesterases was evaluated by monitoring the sensitivity to modification with diethyl pyrocarbonate in the tomato and Aspergillus niger enzymes. Different and incomplete losses of enzyme activity were obtained. Inactivation of the enzymes was proportional to the histidine content (two in the tomato T1 form, six in the Aspergillus form), suggesting that accessible histidine residues do not have active-site functions in these pectinesterases, but contribute to the overall structural stability. Lack of His roles in common between the enzyme forms is in agreement with the structures of pectinesterases having no conserved His residues.

Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons.

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n-acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.

Molecular Mechanisms of Acid Denaturation: The Role of Histidine Residues in the Partial Unfolding of Apomyoglobin

Apomyoglobin adopts a partly folded intermediate conformation (I), sometimes referred to as a molten globule intermediate, near pH 4. To determine which histidine residues trigger this partial unfolding reaction, we made mutants in which nine of the twelve histidine residues in the protein are substituted individually. We then measured acid and urea-induced unfolding curves for these substituted proteins. Two acid unfolding transitions are observed: native (N) to intermediate (I), and I to unfolded (U). These data were fitted using a simple three-state model which has been shown to give an adequate description of acid and urea-induced unfolding of wild-type apomyoglobin. The aim is to quantify changes in the apparent standard Gibbs energy differences between N, I and U, as well as the unfolding mechanism, that result from these substitutions, and to test how well the model fits data for substituted proteins. In most cases, the model fits the data reasonably well, and significant changes in fitted unfolding parameters of various mutants are also clearly visible in the primary data. The following conclusions are drawn. (1) Histidines 24 and 119 synergistically stabilize native apomyoglobin (N) at pH 8, but together destabilize N as pH is decreased below seven. (2) Histidine 36 stabilizes N when it is protonated. (3) Histidine substitutions in the heme-binding pocket (residues 64, 93 and 97) have little effect on the stability of N, suggesting that the heme-binding pocket is open. (4) Histidine substitutions affect the N to I transition but have little effect on the I to U transition. (5) The simple model we use to describe the unfolding of apomyoglobin cannot account for all the data, particularly the effects of the H36Q mutation. The effect of protonated histidine 36 on stabilizing N is not included in the model. We suggest that breaking the hydrogen bond between histidines 24 and 119 by protonation when the pH is decreased from 6 to 4 is an important part of triggering the partial unfolding of N to I, and likewise that formation of the hydrogen bond between histidines 24 and 119 may be a rate-determining step in the kinetic process of forming N from I during refolding.

Role of Histidine Residues in Agonist and Antagonist Binding Sites of A1 Adenosine Receptor

The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N6-[adenine-2,8-3H, ethyl-2-3H]phenylisopropyladenosine ([3H]R-PIA) and antagonist 8-cyclopentyl-1,3-[3H]-dipropylxanthine ([3H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [3H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [3H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A1 adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (KD) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Histidine Residues Are Essential for the Surface Binding and Autoactivation of Human Coagulation Factor XII

The role of histidine residue in the surface binding and autoactivation of human factor XII has been investigated by chemical modification with diethyl pyrocarbonate. It is found that low concentrations of diethyl pyrocarbonate have profound inhibitory effects on the surface binding activity of factor XII. At 2.5-fold molar excess of the reagent, six histidines are modified and 80% of the amidolytic activity is lost. Electrophoretic studies show that the modified protein has lost the capacity to bind to the surface, resulting in diminished proteolytic autoactivation. When modification is performed in the presence of the surface, dextran sulfate, two of the six histidines are protected from modification and the amidolytic activity is completely preserved. It is concluded that histidine residues in factor XII play key role in its surface binding activity.

Role of histidine residues in the α-bungarotoxin binding site of the nicotonic acetylcholine receptor

This paper studies the effect of histidine chemical modification of the membrane-bound acetylcholine receptor from Discopyge tschudii on its specific α-bungarotoxin binding. The acylating reagent ethoxyformic anhydride (diethyl pyrucarbonate, DEP), was used. DEP-treatment induces a loss of binding capacity, time and DEP-concentration dependent. After a 30 min period of derivatization with 2 mM final DEP-concentration, at pH 7.4, the decrease reaches 70%; the loss of binding capacity is faster at pH 7.4 than at pH 6.0, as expected, since the amount of unprotonated species is higher under the first condition. Moreover, when ethoxyformylation is carried out at different pH values, the most important neurotoxin binding decrease occurs between pH 6.0 and 8.0. Furthermore, ethoxyformylation reversion restores such capacity. Consistent with the modification of a binding site, the ethoxyformylation does not bear on the affinity but reduces the number of receptors. Ethoxyformylation in the presence of carbamylcholine shows some ligand protective effect. These results, as a whole, strongly indicate a relevant role for histidine residues at the α-bungarotoxin binding site of the nicotinic acetylcholine receptor.

The effect of histidine modification on the activity of myo-inositol monophosphatase from bovine brain.

The pH dependence of myo-inositol monophosphatase may indicate a role for histidine residues in the catalytic mechanism (Ganzhorn, A. J., and Chanal, M.-C. (1990) Biochemistry 29, 6065-6071). This possibility was investigated by chemical modification. At pH 6.0 and 25 degrees C, the enzyme was inactivated by diethylpyrocarbonate in a pseudo-first order reaction with a bimolecular rate constant of 0.37 M-1 s-1. Two histidines were modified rapidly with no effect on enzyme activity, while 3 residues were modified at a slower rate corresponding to the rate of inactivation. No noticeable changes in the secondary structure of the enzyme were observed by comparison of circular dichroic spectra before and after modification. Treatment of myo-inositol monophosphatase with diethylpyrocarbonate in the presence of inositol 1-phosphate, Mg2+, and Li+ protected 2 residues from modification and decreased the inactivation rate by about 5-fold. Spectrophotometric analysis, the restoration of enzyme activity by hydroxylamine, and the lack of any inhibitory effect with alkylating agents suggest that inactivation is due solely to modification of histidine. We conclude that a histidine residue is essential for activity and may act as a base catalyst during hydrolysis of the substrate.

Role of histidine residues and mechanism of action of isocitrate lyase of castor seedling endosperm

Irradiation of isocitrate lyase of castor seedling endosperm with visible light in the presence of a small concentration of Rose Bengal at pH 6.8 brings about a time-dependent exponential decay of enzyme activity. The first-order rate constant of the inactivation reaction increases with pH in the range pH 6–8 and becomes constant at higher pH values. When the enzyme is treated with diethyl pyrocarbonate (DEPC), a time-dependent exponential decay of its activity is observed and the pseudo first-order rate constant is proportional to the reagent concentration. The loss of activity is accompanied by a parallel increase in absorbance at 240 nm. The latter corresponds to modification of two histidine residues per monomeric subunit of the enzyme for complete inactivation. An analysis of the kinetic data shows that only one of these histidine residues is involved in the catalytic activity. Control experiments ruled out the involvement of residues other than histidine in inactivation by photooxidation and DEPC. Succinate protects the enzyme strongly against inactivation by both procedures but glyoxylate does not. A probable mechanism of action of isocitrate lyase is proposed and discussed in the light of these and earlier results.

Site‐directed mutagenesis at histidines of aerolysin from Aeromonas hydrophila: a lipid planar bilayer study

The role of histidine residues in the formation of channels by the cytolytic toxin aerolysin has been studied in planar lipid bilayers by substituting each of the six histidines in the native protein with asparagine. His341 or His186 mutants had the same channel-forming ability as native toxin, whereas the His332 and His121 mutants were less active. Mutations at His132 and His107, which interfere with the oligomerization of the toxin, drastically reduce pore formation. These findings support the conclusion that oligomerization of the toxin must precede channel formation, and that at least two of the six histidine residues are essential for this to occur. The aerolysin channel is a water-filled pore with an approximate diameter of 9.3 +/- 0.4 A.

The Role of Histidine Residues in the Non-Enzyme Covalent Attachment of Glucose and Ascorbic Acid to Protein

Copper ions have been suggested to play a role in the non-covalent glycosylation (glycation) of proteins via transition metal-catalysed oxidations. We have further investigated "autoxidative glycosylation" by comparison of the behaviour of dog and bovine serum albumin with respect to the oxidative reactions of glucose and ascorbate. The proteins possess similar numbers of total amino residues available for glucose attachment but dog serum albumin contains fewer histidine groups and also lacks a high affinity copper-binding site. We find that the higher copper-binding capacity of bovine serum albumin is reflected in a lower rate of ascorbate oxidation as well as less protein oxidative damage than is the case for dog serum albumin. We also observe that modification of bovine serum albumin histidine groups by diethylpyrocarbonate enhances ascorbate-mediated protein fluorophore formation.

On the role of histidine residues in cyclodextrin glycosyltransferase: chemical modification with diethyl pyrocarbonate

Ethoxyformylation with diethyl pyrocarbonate of ∼ 1.5 His residues per molecule of enzyme reduced the cyclising activity of both the α-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain M 5 al and the β-cyclodextrin glycosyltransferase from Bacillus circulans strain 8 by >90%. Pre-incubation with substrate protected the enzymes from ethoxyformylation. Digestion of starch by the modified enzymes resulted in a delayed formation of cyclodextrins (cyclomalto-oligosaccharides, CDs), but a marked increase in the production of reducing saccharides. Similarly, coupling of αCD and maltose and successive disproportionation yielded mainly glucose and malto-oligosaccharides. The results are discussed in the context of the role of conserved His residues for binding of substrate and the transfer reactions.

The role of histidine residues in the alpha toxin of Clostridium perfringens

The role of histidine residues in the alpha toxin of Clostridium perfringens

Role of histidine residues in the binding mechanism of the growth hormone family

1. Reactivity of the hGH histidine residues were studied by reaction with ethoxyformic anhydride. Localization in the molecule of three kinetically distinguishable classes, each including only one residue, was achieved. 2. The first was composed of residue 151, with an apparent velocity constant k = 0.735/min, (similar to that of histidines 19 and 21 in bGH and eGH). The second histidine, 18, with a velocity constant k = 0.135/min, (similar to that of histidine 169 in the above hormones), and a third, histidine 21, which does not react at all. 3. Neither histidine 151 nor 18 seem to be involved, at least not directly, in bGH binding to specific rat liver sites, since the decrease in this capacity was only 47% after modification of the former by 77 and 65% after total modification of the latter. 4. These results, and those previously obtained with bGH and eGH, suggest that either histidine 21 is the only indispensable histidine for the binding of growth hormones to specific rat liver sites, or that histidine 21 and/or 18 (19 in bGH and eGH), are located within the growth hormone binding site interaction area.

The cytochrome c peroxidase-cytochrome c electron transfer complex. The role of histidine residues.

The histidine-selective reagent diethyl pyrocarbonate and dye-sensitized photooxidation have been used to study the functional role of histidines in cytochrome c peroxidase. Of the 6 histidines in cytochrome c peroxidase, 5 are modified by diethyl pyrocarbonate at alkaline pH and 4 by photooxidation. The sixth histidine serves as the proximal heme ligand and is unavailable for reaction. Both modification reactions result in the loss of enzymic activity. However, photooxidized peroxidase retains its ability to react with H2O2 and to form a 1:1 cytochrome c peroxidase-cytochrome c complex. It is, therefore, concluded that the extra histidine modified by diethyl pyrocarbonate is the catalytic site distal histidine, His 52. In the presence of cytochrome c, no enzymic activity is lost by photooxidation and a single histidine, His 181, is protected from oxidative destruction. This finding provides strong support for the hypothetical model of the cytochrome c peroxidase-cytochrome c complex in which His 181 lies near the center of the intermolecular interface where it seems to provide an important link in the electron transfer process.

Multifunctionality of lipoamide dehydrogenase: Role of histidine residues in reductase reaction

Rose bengal sensitizes photoinactivation of lipoamide dehydrogenase from pig heart to a constant residual reductase activity resulting from specific destruction of histidine residues. The rate of sensitized photoinactivation is pH dependent and is associated with an ionizable group with p K 6.6 ± 0.2. All steady-state kinetic parameters are markedly reduced by photooxidation. Spectroscopic studies indicate the contribution of oxidized flavin/dithiol to the half-reduced form of the photooxidized enzyme. The proton magnetic resonance spectrum of lipoamide dehydrogenase shows resolved histidine C 2 proton peak at δ9.18 ppm and a shoulder at δ9.23 ppm. The shoulder protons are eliminated by the sensitized photooxidation and shifted upfield on deprotonation. At high pH, the characteristic Faraday A term also disappears. These observations suggest that the essential histidine stabilizes the nascent thiolate via the ion pair formation to facilitate the reductase reaction catalyzed by lipoamide dehydrogenase.