Interaction between azo dye Acid Red 14 and pepsin by multispectral methods and docking studies

The interaction of synthetic azo dye Acid Red 14 with pepsin was studied by fluorescence spectroscopy, UV–vis spectroscopy, circular dichroism and molecular docking. Results from the fluorescence spectroscopy show that Acid Red 14 has a strong capability to quench the intrinsic fluorescence of pepsin with static quenching. Binding constant, number of the binding sites and thermodynamic parameters were measured at different temperatures. The result indicates that Acid Red 14 interact with pepsin spontaneously by hydrogen bonding and van der Waals interactions. Three‐dimensional fluorescence spectra and circular dichroism spectra reveal that Acid Red 14 could slightly change the structure of pepsin. The hydrogen bond is formed between Acid Red 14 and Tyr‐189 and Thr‐218 residues of pepsin. Furthermore, the binding between Acid Red 14 and pepsin inhibits pepsin activity. The study can provide a way to analyze the biological safety of Acid Red 14 on digestive proteases or other proteins.     

Conformation of pepsin and pepsinogen: some aspects of the role of tyrosine residues and the 1-44 segment of pepsinogen on conformational stability

Conformational changes induced in pepsin and pepsinogen by iodination of tyrosine residues and the possible role of lysine residues on conformational stability of pepsinogen are investigated by circular dichroism (CD) studies in solution. At low degrees of iodination (6 I/molecule) the pepsin molecule denatured, with complete loss of β-structure at pH 5.5. Pepsinogen showed greater resistance to conformational change on iodination (10 I/molecule) and about 30% of its ordered structure is retained. In the aromatic region, the tyrosyl CD bands of iodinated pepsin decreased in intensity, indicating a change in the environment of tyrosine residues. A comparison with the CD spectra of expanded structures of pepsin in 6 m guanidine hydrochloride or alkaline solutions (pH 9.75) indicated retention of a significant amount of tertiary structure in iodinated pepsin. Changes in tertiary structures were marginal on iodination of pepsinogen. Less than 1% (residue moles) of poly-l-lysine, a known inhibitor, was found to destabilize the secondary and tertiary structure of pepsin at pH 6.75, although the lysine-rich 1–44 segment of pepsinogen tends to stabilize the conformation of the pepsin chain. This seems to suggest that the inhibitory effects of polylysine on pepsin occur by a mechanism different from that of the activity-limiting effect of the lysine-rich 1–44 segment of pepsinogen.     

Stability of RNA hairpin loops: A 6 -C m -U 6

The thermodynamics and circular dichroism of a series of A₆-C_m-U₆ (m = 4, 5, 6 or 8) oligoribonucleotides have been studied. These molecules form intramolecular hairpin loops at low temperatures and therefore are useful models for similar structures which occur in larger, natural RNA molecules. The stability of the helix forming the stem of these loops was found to be considerably greater than an intermolecular helix with the same length and composition. The most stable loop is m = 6. The enthalpy for initiation of the loop is unfavorable; it ranges from + 24 kcal, for m = 4 to + 21 kcal, for m = 6. The maximum in stability for the C₆ loop and the large positive enthalpy for loop initiation are in disagreement with expectations from simple theories assuming a Gaussian distribution of end-to-end distances. Loop strain for m = 4 and m = 5 and the unstacking of the cytosines on loop formation are likely physical explanations for these thermodynamic data. The circular dichroism spectrum of cytosine residues in the C₆ and C₈ loops is very similar to the spectrum of single-stranded oligoribocytidylate. However, the cytosine residues in the C₅ loop have a very different circular dichroism spectrum from the corresponding oligo(C₅) spectrum. In accordance with the thermodynamic data, we conclude from the circular dichroism data that the C₅ loop has an altered conformation from the C₅ and C₈ loops.     

Revised 2.3 A structure of porcine pepsin: evidence for a flexible subdomain

A revised three-dimensional crystal structure of ethanol-inhibited porcine pepsin refined to an R-factor of 0.171 at 2.3 A resolution is presented and compared to the refined structures of the fungal aspartic proteinases: penicillopepsin, rhizopuspepsin, and endothiapepsin. Pepsin is composed of two nearly equal N and C domains related by an intra dyad. The overall polypeptide fold and active site structures are homologous for pepsin and the fungal enzymes. The weak inhibition of pepsin by ethanol can be explained by the presence of one or more ethanol molecules, in the vicinity of the active site carboxylates, which slightly alter the hydrogen-bonding network and which may compete with substrate binding in the active site. Structural superposition analysis showed that the N domains aligned better than the C-domains for pepsin and the fungal aspartic proteinases: 107-140 C alpha pairs aligned to 0.72-0.85 A rms for the N domains; 64-95 C alpha pairs aligned to 0.78-1.03 A rms for the C domains. The major structural difference between pepsin and the fungal enzymes concerns a newly described subdomain whose conformation varies markedly among these enzyme structures. The subdomain in pepsin comprises nearly 100 residues and is composed of two contiguous segments within the C domain (residues 192-212 and 223-299). the subdomain is connected, or "hinged," to a mixed beta-sheet that forms one of the structurally invariant, active site psi-loops. Relative subdomain displacements as large as a 21.0 degrees rotation and a 5.9 A translation were observed among the different enzymes. There is some suggestion in pepsin that the subdomain may be flexible and perhaps plays a structural role in mediating substrate binding, determining the substrate specificity, or in the activation of the zymogen.     

Structural characterization of myo-inositol monophosphatase from bovine brain by secondary structure prediction,fluorescence, circular dichroism and Raman spectroscopy

Structural aspects of myo-inositol monophosphatase were examined by spectroscopic techniques and empirical prediction methods. The enzyme belongs to the α/β class of proteins, with approx. 33% α-helix and 29% β-sheet, as shown by circular dichroism (CD), Raman spectroscopy and prediction based on the amino-acid sequence. The Raman spectrum also suggests that the three tryptophan residues in myo-inositol monophosphatase are not expose to solvent. This was confirmed by a blue shift of 25 nm in the fluorescence emission spectrum, as compared to tryptophan in water, and by quenching studies with acrylamide. The enzyme shows a transition temperature of 87°C for the CD signal at 222 nm. This remarkable heat stability is not due to the presence of disulfide bonds, since both the Raman spectrum and chemical modification studies clearly indicate that all six cysteine residues are in the reduced state.     

A spectroscopic and conformational study of pertussis toxin

The conformation of native pertussis toxin has been investigated by secondary structure prediction and by circular dichroism, fluorescence and second-derivative ultraviolet absorption spectroscopy. The far-ultraviolet circular dichroic spectrum is characteristic of a protein of high beta-sheet and low alpha-helix content. This is also shown by an analysis of the circular dichroic spectrum with the Contin programme which indicates that the toxin possesses 53% beta-sheet, 10% alpha-helix and 37% beta-turn/loop secondary structure. Second-derivative ultraviolet absorption spectroscopy suggests that 34 tyrosine residues are solvent-exposed and quenching of tryptophan fluorescence emission has shown that 4 tryptophan residues are accessible to iodide ions. One of these tryptophans appears to be in close proximity to a positively charged side-chain, since only 3 tryptophans are accessible to caesium ion fluorescence quenching. When excited at 280 nm, the emission spectrum contains a significant contribution from tyrosine fluorescence, which may be a consequence of the high proportion (55%) of surface-exposed tyrosines. No changes in the circular dichroic spectra of the toxin were found in the presence of the substrate NAD. However, NAD did quench both tyrosine and tryptophan fluorescence emission but did not change the shape of the emission spectrum, or the accessibility of the tryptophans to either the ionic fluorescence quenchers or the neutral quencher acrylamide.     

Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3

The three-dimensional structures of pepsin inhibitor-3 (PI-3) from Ascaris suum and of the complex between PI-3 and porcine pepsin at 1. 75 A and 2.45 A resolution, respectively, have revealed the mechanism of aspartic protease inhibition by this unique inhibitor. PI-3 has a new fold consisting of two domains, each comprising an antiparallel beta-sheet flanked by an alpha-helix. In the enzyme-inhibitor complex, the N-terminal beta-strand of PI-3 pairs with one strand of the 'active site flap' (residues 70-82) of pepsin, thus forming an eight-stranded beta-sheet that spans the two proteins. PI-3 has a novel mode of inhibition, using its N-terminal residues to occupy and therefore block the first three binding pockets in pepsin for substrate residues C-terminal to the scissile bond (S1'-S3'). The molecular structure of the pepsin-PI-3 complex suggests new avenues for the rational design of proteinaceous aspartic proteinase inhibitors.     

Studies on the Mechanism of Activation of Pepsinogen

Experiments were carried out on the effects of substrate or competitive inhibitor on the rate of appearance of N-terminal isoleucine residue of pepsin and peptides released from pepsinogen in its conversion to pepsin. Assumptions were made from these experiments, that an active site is initially formed in pepsinogen by acidification of its solution, and that peptide bond between 41-glutamyl and 42-isoleucyl residues locates in the juxtaposition to the active site forming an intramolecular enzyme-substrate complex. Thus, N-terminal tail of pepsinogen is released by a hydrolysis catalyzed by its own active site.

It was Indeed ascertained in this study that neither a small amount of pepsin which could be accompanied by pepsinogen preparation used contributes to the initial step of hydrolysis of pepsinogen nor pepsin formed accelerates the following activation process.

Therefore, it was concluded that the conversion of pepsinogen to pepsin is self-degrad-ation process.     

Effect of dimethyl sulphoxide on the crystal structure of porcine pepsin

The structure of porcine pepsin crystallized in the presence of dimethyl sulphoxide has been analysed by X-ray crystallography to obtain insights into the structural events that occur at the onset of chemical denaturation of proteins. The results show that one dimethyl sulphoxide molecule occupies a site on the surface of pepsin interacting with two of its residues. An increase in the average temperature factor of pepsin in the presence of dimethyl sulphoxide has been observed indicating protein destabilization induced by the denaturant. Significant increase in the temperature factor and weakening of the electron density have been observed for the catalytic water molecule located between the active aspartates. The conformation of pepsin remains unchanged in the crystal structure. However, the enzyme assay and circular dichroism studies indicate that dimethyl sulphoxide causes a slight change in the secondary structure and complete loss of activity of pepsin in solution.     

A new fluorescent microassay method for pepsin using succinyl-albumin.

A method was developed for fluorescent microassay of pepsin with a fluorescent reagent, fluorescamine, and a nonquenching substrate, succinyl-albumin. In this method hydrolysis of succinyl-albumin by pepsin at pH 2,0 was stopped by adding phosphate buffer, pH 6.1, and newly liberated amino groups in the reaction mixture were determined quantitatively by fluorescence after adding fluorescamine. Fluorescence increased linearly with 1.0 to 18 ng of hog pepsin. The assay was 200 times more sensitive than the modified micromethod of Anson [(1939) J. Gen. Phys.22, 79–89].     

Molecular and crystal structures of monoclinic porcine pepsin refined at 1.8 A resolution

The molecular structure of the archetypal aspartic proteinase, porcine pepsin (EC 3.4.23.1), has been refined using data collected from a single monoclinic crystal on a twin multiwire detector system to 1.8 A resolution. The current crystallographic R-factor (= sigma parallel to Fo/-/Fc parallel to/sigma/Fo/) is 0.174 for the 20,519 reflections with /Fo/ greater than or equal to 3 sigma (Fo) in the range 8.0 to 1.8 A (/Fo/ and /Fc/ are the observed and calculated structure factor amplitudes respectively). The refinement has shown conclusively that there are only 326 amino acid residues in porcine pepsin. Ile230 is not present in the molecule. The two catalytic residues Asp32 and Asp215 have dispositions in porcine pepsin very similar to the dispositions of the equivalent residues in the other aspartic proteinases of known structure. A bound solvent molecule is associated with both carboxyl groups at the active site. No bound ethanol molecule could be identified conclusively in the structure. The average thermal motion parameter of the residues that comprise the C-terminal domain of pepsin is approximately twice that of the residues in the N-terminal domain. Comparisons of the tertiary structure of pepsin with porcine pepsinogen, penicillopepsin, rhizopus pepsin and endothia pepsin reveal that the N-terminal domains are topographically more similar than the conformationally flexible C-terminal domains. The conformational differences may be modeled as rigid-body movements of "reduced" C-terminal domains (residues 193 to 212 and 223 to 298 in pepsin numbering). A similar movement of the C-terminal domain of endothia pepsin has been observed upon inhibitor binding. A phosphoryl group covalently attached to Ser68 O gamma has been identified in the electron density map of porcine pepsin. The low pKa1 value for this group, coupled with unusual microenvironments for several of the aspartyl carboxylate groups, ensures a net negative charge on porcine pepsin in a strongly acid medium. Thus, there is a structural explanation for the very early observations of "anodic migration" of porcine pepsin at pH 1. In the crystals, the molecules are packed tightly into a monoclinic unit cell. There are 190 direct contacts (less than or equal to 4.0 A) between a central pepsin molecule and the five unique symmetry-related molecules surrounding it in the crystalline lattice. The tight packing in this cell makes pepsin's active site and binding cleft relatively inaccessible to substrate analogs or inhibitors.     

N-terminal modifications increase the neutral-pH stability of pepsin

A structure-function study was undertaken to determine the effects of N-terminal mutations in pepsin designed to introduce the Lys-X-Tyr motif and increase N-terminal flexibility. At pH 7.0, E7K/T12A/E13Q pepsin was inactivated more slowly compared to WT, whereas the mutants E7K and T12A/E13Q were not stabilized. Far-UV circular dichroism revealed that changes in secondary structure accompanied the inactivation process, and that the structural changes occurred at approximately the same rate as inactivation. All of the inactivated pepsin forms showed retention of substantial secondary structure, more than previously determined for pepsin denatured at pH 7.2 and 8.0, suggesting the presence of a structural intermediate at pH 7.0. The coupled mutations at positions 12 and 13 impacted the pH dependence of activity at pH 0.9, lowered affinity for a synthetic substrate, and lowered the turnover number. The introduction of Lys at position 7 apparently destabilized the interaction between prosegment-enzyme body as evidenced by activation at higher pH (>or= 4.0) compared to WT, but showed no change for pH dependence of activity, nor a statistically significant change in affinity for the synthetic substrate.     

Circular dichroism and laser Raman spectroscopic analysis of the secondary structure ofCerebratulus lacteus toxin B-IV

The secondary structure ofCerebratulus lacteus toxin B-IV, a neurotoxic polypeptide containing 55 amino acid residues and four disulfide bonds, was experimentally estimated by computer analyses of toxin circular dichroism (CD) and laser Raman spectra. The CD spectrum of the toxin displayed typical α-helical peaks at 191, 208, and 222 nm. At neutralpH, the α-helix estimates from CD varied between 49 and 55%, when nonrepresentative spectrum analytical methods were used. Analysis of the laser Raman spectrum obtained at a much higher toxin concentration yielded a 78% α-helix estimate. Both CD and Raman spectroscopic methods failed to detect any β-sheet structure. The spectroscopic analyses revealed significantly more α-helix and less β-sheet for toxin B-IV than was predicted from its sequence. To account for the difference between the 49–55% helix estimate from CD spectra and the 78% helix estimate from the Raman spectrum, we postulate that some terminal residues are unfolded at the low toxin concentrations used for CD measurements but form helix at the high toxin concentration used for Raman measurements. Our CD observations showing thatCerebatulus toxin B-IV helix content increases about 15% in trifluoroethanol or at highpH are consistent with this interpretation.     

Cleavage specificities of aspartic proteinases toward oxidized insulin B chain at different pH values

The cleavage specificities of typical aspartic proteinases: pepsin A, gastricsin, cathepsin D and rhizopuspepsin, were examined at different pH values with oxidized insulin B chain as a substrate with special attention to the specificities near neutral pH. Significant differences in relative specificity for scissile bonds were observed between pH 2.0 and 5.5-6.5, which may be partly related with the changes in dissociation states of the His and Glu residues in the substrate and the ionizable residues in the active site of each enzyme.     

The specific modification of histidyl residues of inorganic pyrophosphatase from Bacillus stearothermophilus by photooxidation

Photooxidation of inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] from Bacillus stearothermophilus in the presence of rose bengal resulted in rapid loss of enzymatic activity. The pH profile of the inactivation rate by the photooxidation showed an inflection point around pH 6.8, suggesting the involvement of histidyl residues in the inactivation. Amino acid analysis revealed that the loss of enzymatic activity was accompanied by the destruction of 3 histidyl residues per molecule. The presence of Mg2+ alone afforded partial protection against the inactivation, whereas inorganic pyrophosphate, the substrate, showed almost no protective effect against inactivation. The photooxidation of inorganic pyrophosphatase altered the circular dichroism spectrum and the difference UV spectrum induced by Mg2+ in the near ultraviolet region. These results suggested that histidyl residues appear to be located at the binding site of Mg2+ and may contribute to the conformational change induced by Mg2+.     

Malonichrome,a new iron chelate from Fusarium roseum

The predominant iron chelates, or siderochromes, produced by the fungus, Fusarium roseum during culture periods up to seven days are the ester type fusarinine compounds. During longer periods of incubation, the fusarinine compounds completely disappear from the culture medium and are replaced by a new siderochrome. The new compound has been isolated, purified, and its structure determined. It is a cyclic hexapeptide containing one residue of l-alanine, two residues of glycine and three residues of δ-N-hydroxyornithine. The hydroxylamino groups of the ornithine residues are acylated with 3 mol of malonic acid to form a negatively charged ferrichrome type chelate. The circular dichroism spectrum indicates that the stereochemistry about the iron is Λ-cis. This compound, which we name malonichrome, is not an efficient iron donor to F. roseum nor does it show growth factor activity towards Arthrobacter flavescens.     

Comparative specificity of microbial acid proteinases for synthetic peptides. Primary specificity with Z-tetrapeptides

The substrates Z-X

Leu-(Ala)₂ and

Z-Phe X-(Ala)₂ (Z = benzyloxycarbonyl, X = various amino acid residues) were synthesized in order to investigate the primary specificity of acid proteinases from molds and yeasts. Since these peptides are mainly susceptible to cleavage by the enzymes at the peptide bonds shown by the arrows, it was possible to determine the specificity with respect to the amino acid residues on both sides of the splitting point. Pepsin was used for comparison. The results indicated that the microbial acid proteinases exhibit specificity for aromatic or hydrophobic amino acid residues on both sides of splitting point in peptide substrates, as does pepsin. However, the microbial enzymes showed somewhat broader specificity than pepsin. The former enzymes, which possess trypsinogen-activating ability, show specificity for a lysine residue, while pepsin or Mucor rennin-like enzyme does not. Although pepsin is very specific for a tyrosine residue on the imino side of the splitting point, the microbial enzymes do not show such stringency.     

Understanding the Mechanism of Prosegment-catalyzed Folding by Solution NMR Spectroscopy

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.     

Multi-spectroscopic studies on the interaction between traditional Chinese herb,helicid with pepsin

Study on the binding properties of helicid by pepsin systematically using multi-spectroscopic techniques and molecular docking method, and these interactions comprise biological recognition at molecular level and backbone of biological significance in medicine concerned with the uses, effects, and modes of action of drugs. We investigated the mechanism of interaction between helicid and pepsin by using various spectroscopic techniques viz., fluorescence spectra, UV–Vis absorption spectra, circular dichroism (CD), 3D spectra, synchronous fluorescence spectra and molecular docking methods. The quenching mechanism associated with the helicid–pepsin interaction was determined by performing fluorescence measurements at different temperatures. From the experimental results show that helicid quenched the fluorescence intensity of pepsin via a combination of static and dynamic quenching process. The binding constants (K_a) at three temperatures (288, 298, and 308 K) were 7.940?×?10⁷, 2.082?×?10⁵ and 3.199?×?10⁵ L mol^?1, respectively, and the number of binding sites (n) were 1.44, 1.14, and 1.18, respectively. The n value is close to unity, which means that there is only one independent class of binding site on pepsin for helicid. Thermodynamic parameters at 298 K were calculated as follows: ΔH^o (??83.85 kJ mol^?1), ΔG^o (??33.279 kJ mol^?1), and ΔS^o (??169.72 J K^?1 mol^?1). Based on thermodynamic analysis, the interaction of helicid with pepsin is driven by enthalpy, and Van der Waals’ forces and hydrogen bonds are the main forces between helicid and pepsin. A molecular docking study further confirmed the binding mode obtained by the experimental studies. The conformational changes in the structure of pepsin was confirmed by 3D fluorescence spectra and circular dichroism.     

Roles of Tyr13 and Phe219 in the unique substrate specificity of pepsin B

Pepsin B is known to be distributed throughout mammalia, including carnivores. In this study, the proteolytic specificity of canine pepsin B was clarified with 2 protein substrates and 37 synthetic octapeptides and compared with that of human pepsin A. Pepsin B efficiently hydrolyzed gelatin but very poorly hydrolized hemoglobin. It was active against only a group of octapeptides with Gly at P2, such as KPAGF/LRL and KPEGF/LRL (arrows indicate cleavage sites). In contrast, pepsin A hydrolyzed hemoglobin but not gelatin and showed high activity against various types of octapeptides, such as KPAEF/FRL and KPAEF/LRL. The specificity of pepsin B is unique among pepsins, and thus, the enzyme provides a suitable model for analyzing the structure and function of pepsins and related aspartic proteinases. Because Tyr13 and Phe219 in/around the S2 subsites (Glu/Ala13 and Ser219 are common in most pepsins) appeared to be involved in the specificity of pepsin B, site-directed mutagenesis was undertaken to replace large aromatic residues with small residues and vice versa. The Tyr13Ala/Phe219Ser double mutant of pepsin B was found to demonstrate broad activity against hemoglobin and various octapeptides, whereas the reverse mutant of pepsin A had significantly decreased activity. According to molecular modeling of pepsin B, Tyr13 OH narrows the substrate-binding space and a peptide with Gly at P2 might be preferentially accommodated because of its high flexibility. The hydroxyl can also make a hydrogen bond with nitrogen of a P3 residue and fix the substrate main chain to the active site, thus restricting the flexibility of the main chain and strengthening preferential accommodation of Gly at P2. The phenyl moiety of Phe219 is bulky and narrows the S2 substrate space, which also leads to a preference for Gly at P2, while lowering the catalytic activity against other peptide types without making a hydrogen-bonding network in the active site.