Aspartic proteinases: Fourier transform infrared spectroscopic studies of a model of the active side.

We synthesized and studied by Fourier transform infrared spectroscopy nine monosalts of diamides as models for the active side of aspartic proteinases. One compound, the monosalt of meta-aminobenzoic acid diamide of fumaric acid (m-FUM), shows the same biological activity as pepsin with regard to the splitting of peptide bonds of the Pro-Thi-Glu-Phe-Phe(4-NO2)-Arg-Leu heptapeptide. The monosalt of m-FUM forms with oxindole a complex in which the carboxylic acid group of the monosalt of m-FUM is strongly hydrogen bonded with the O atom of the peptide bond of oxindole. When one water molecule is added to this complex, the strong field of the carboxylate group destabilizes an O-H bond of the water molecule. The distorted water molecule attacks the carbon atom of the peptide group, and the water proton transfers to the peptide N atom. Simultaneously, the C-N bond of the amide group is broken. Hence it is demonstrated that the catalytic mechanism of aspartic acid proteinases is a base catalysis. The results show that for this catalytic mechanism there are sufficient carboxylic and carboxylate groups, as well as a water molecule in the correct arrangement. It was also demonstrated with other monosalts of dicarboxylic acids that well-defined steric conditions of the carboxylic acid and the carboxylate group must be fulfilled to show hydrolytic activity with regard to oxindole molecules.     

Interactions of nicotinamide-adenine dinucleotide phosphate analogues and fragments with pigeon liver malic enzyme. Synergistic effect between the nicotinamide and adenine moieties.

The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5'-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3'-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2'-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2'-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.     

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. IV. Structure around the Sa thiol group.

The structure of a tryptic peptide containing one specific sulfhydryl group (Sa), which is responsible for the activation of Mg2+-ATPase of myosin B and is present in the light meromyosin region of the myosin molecule, was studied. The amino acid sequence was deduced to be Thr (or Ser)-Asn-Ala-Ala-Cys-Ala-Ala-Leu-Asp-Lys-Lys. In addition, a space-filling model around Sa was built up by comparing Sa-peptide with the amino acid sequence around Cys 190 of alpha-tropomyosin, and the high reactivity of Sa with N-ethylmaleimide is considered based on this model.     

Kinetic studies of the alkaline mesentericopeptidase. Study on the active centre topography of alkaline mesentericopeptidase by means of bifunctional reversible inhibition.

The inhibitory effect of alkylboronic acids H(CH2)nB(OH)2(n=2-8) and Ph(CH2)n-B(OH)2, (n=0-4), on the alkaline mesentericopeptidase-catalysed hydrolysis of synthetic substrates was studied. It was shown that alkylboronic acids act as bifunctional reversible inhibitors. The borate group interacts with an ionogenic group of the enzyme with a pKa of about 6.9-7.0. The latter is probably the catalytically active imidazole of the active centre. The hydrocarbon part of the molecule also takes part in the formation of the enzyme-inhibitor complex. The dependence of the degree of the enzyme-inhibitor complex formation upon the length of the side-chain of the inhibitor indicates the presence of two binding sites on the enzyme molecule.     

Interaction of topotecan--a DNA topoisomerase inhibitor--with dual-stranded polydeoxyribonucleotides. I. Dimerization of topotecan in solution]

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5-11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa = 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10(-5) M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10(-6) M) to 7.1 ([TPT] = 10(-4) M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 +/- 0.7) x 10(3) M-1.     

The reaction of penicillin with proteins.

The mode of reaction of benzylpenicillin with two proteins was studied, with particular reference to the allergenicity of penicillin. These reactions, with pig insulin, and with hen's-egg-white lysozyme, were carried out in neutral solution at 37 degrees C. High concentrations of penicillin are needed to label the proteins, owing to concurrent hydrolysis of penicillin. Evidence has been obtained that the penicillin-reactive sites on the insulin molecule are the alpha-amino group at the N-terminus of the A chain and the epsilon-amino group of the lysine residue; whereas a site of reaction with lysozyme appears to be the epsilon-amino group of lysine-116.     

Radical formation in pyrimidines. IV. 1,3-dimethyluracil, a non-hydrogen-bonding crystal.

Radical formation in single crystals of 1,3-dimethyluracil by X-irradiation has been studied by electron spin resonance at 9.5 GHz. This crystal contains no hydrogen bonds. Only Van der Waals forces are present. Accordingly, after X-irradiation at 300 K, the only radicals observed are those resulting from the excitation path: the H-addition radical at C5 and an H-abstraction radical from a methyl group. Irradiation with light of lambda more than 400 nm induces the transformation of the C5-addition into the C6-addition radical. INDO calculations indicate that the C6-addition radical is protonated at O4. Since this crystal does not contain N-H or O-H bonds, this protonation can only occur through proton-abstraction from a C-H bond of a neighbouring molecule by the carbonyl group. The presence of short contacts between C6 and O4 is taken to suggest that the abstraction occurs at C6.     

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.     

A 1H nuclear relaxation study of the Mn2+ . bleomycin complex. Proximity of the metal to the DNA-binding site

The antineoplastic action of bleomycin is thought to involve the aerobic degradation of DNA by the Fe2+ . bleomycin complex. Different parts of the bleomycin molecule have been implicated in metal binding and DNA binding. To probe the structure of a metal-containing bleomycin, we studied the effects of the high spin Mn2+ ion in the Mn2+ . bleomycin complex on the longitudinal nuclear relaxation rates of various protons in the molecule. Complexation of Mn2+ to bleomycin was also studied by EPR, and a Scatchard plot of the EPR data revealed a single tight divalent cation-binding site per molecule. From the magnitudes of the paramagnetic effects of Mn2+ on the nuclear relaxation rates of several assigned resonances, we calculate the relative distances of the corresponding protons from the metal. Using a pyrimidine methyl to metal distance of 6.5 A, consistent with the metal coordination of this aromatic group of bleomycin established on the basis of other studies, we find from our data that the bithiazole and COOH-terminal portions of the molecule are located spatially very close to the metal. These groups have previously been implicated in DNA binding. Our metal to bithiazole proton distances (approximately 5.4 A) are consistent with bithiazole as a metal ligand, although possible involvement of interactions other than direct coordination in maintaining close proximity cannot be excluded. Our distance data also argue against the imidazole ring of beta-hydroxyhistidine as a ligand. The short distance between the metal- and DNA-binding sites indicated by our studies would help ensure that the reactive reduced oxygen radicals produced at the metal site during Fe2+ oxidation in the aerobic Fe2+ . bleomycin complex reach the substrate DNA before the destruction of these radicals can occur in other ways.     

Electron microscopy and image analysis of the GroEL-like protein and its complexes with glutamine synthetase from pea leaves.

The molecular structure of GroEL-like protein from pea leaves has been studied by electron microscopy and image analysis of negatively stained particles. Over 1500 molecular projections were selected and classified by multivariate statistical analysis. It was shown that the molecule consists of 14 subunits arranged in two layers with 72 point group symmetry. Side view projections of the molecule show a four-striation appearance, which subdivides both layers of seven subunits into two halves; this may be explained by a two-domain structure of the subunits. The presence in protein preparations of projections corresponding to one layer of subunits or half-molecules is consistent with the molecular structure suggested. Electron microscopic evidence for a specific association of GroEL-like protein and octameric glutamine synthetase, which was co-purified with this protein, was obtained.     

Secondary electron transfer in reaction centers of Rhodopseudomonas sphaeroides. Out-of-phase periodicity of two for the formation of ubisemiquinone and fully reduced ubiquinone.

Electron transfer between purified reaction centers from Rhodopseudomonas sphaeroides and exogenous ubiquinone has been studied in the presence of electron donors by measurements of light-induced absorbance changes following a sequence of short actinic light flashes. Each odd flash promotes the formation of a molecule of ubisemiquinone; after each even flash the semiquinone disappears and a molecule of the fully reduced quinone appears. We interpret these results by means of a model where a specialized molecule of ubiquinone is reduced by the primary electron acceptor in a one-electron transfer reaction after each flash, and is reoxidized by a molecule of the ubiquinone pool in a two-electron transfer reaction every two flashes.     

The unreliability of estimates of group dissociation constants.

If a molecule contains two groups that bind a common ligand, then to determine their group dissociation constants it is necessary (1) to assume that some property of the molecule is affected by the degree of ligation of only one of the groups, and (2) to determine how this property varies with the concentration of free ligand. When assignment of group dissociation constants made on this basis suggests that the degree of ligation of each group greatly affects the dissociation constant of the other, then the original assumption that the chosen property is completely unaffected by ligation of the second group is rendered doubtful. Dissociation constants of interacting groups can therefore not be reliably determined.     

Theoretical study of conformational flexibility of tuftsin in vacuum and in aqueous environment.

Conformational flexibility of tuftsin molecule is studied using all-atom based atom-atom potential and systematic search, simulated annealing molecular dynamics (SAMD) and molecular dynamics (MD) techniques. Latter was carried out for 650 pico seconds (ps) using AMBER 4.0 with explicit water in TIP3P model. Number of inter-atomic distances and torsional angles were monitored during SAMD and MD simulation. We found that tuftsin molecule, irrespective of any starting conformation, assumes highly folded structure with strong electrostatic interaction between Lys-2 NH3 and Arg-4 carboxylic group and weak hydrogen bond between Lys-2 CO and Arg-4 NH atoms. It had distorted but stable conformation close to inverse gamma turn.     

Chloramine-T in radiolabeling techniques. III. Radioiodination of biomolecules containing thioether groups

A previously reported method for iodination of the tyrosine moiety of oxidation-sensitive biomolecules was found to cause unacceptable damage to biomolecules containing thiols and thioether groups. This was due to the oxidation of the sulfur-containing residues by molecular iodine (I(2)). To selectively iodinate the tyrosine moiety with minimum oxidation to the sulfur functionality, studies of the kinetics of the reactions between I-(3) and various amino acids and small peptides at various pH values in phosphate buffer were undertaken. Within the pH range studied (5.5-8.2), the results showed that the iodination reaction is strongly catalyzed by hydroxide ions, whereas the oxidation of the sulfur group was insensitive to pH. The results also showed that both reactions are strongly catalyzed by HPO-(4) ion. In a complex molecule, such as methionine-enkephalin, oxidation of the methionine residue (undesirable reaction) proceeds in parallel with iodination of the tyrosine residue (desirable reaction). If such a molecule was iodinated in 0.01 M phosphate buffer at pH values above 7.5, the iodination reaction would proceed much more rapidly than the oxidation reaction, resulting in a high yield of iodinated substrate with little oxidative damage.     

Solubility of phosphatidylcholine in chloroform. Formation of hydrogen bonding between phosphatidylcholine and chloroform

The solubility of phosphatidylcholine (PC) was studied by the spectroscopic analysis and the measurement of the solubility. The qualitative analysis of infrared absorptionspectra confirmed the existence of two types of hydrogen bondings between chloroform and PC, one between chloroform and the CO group of PC and the other between chloroform and the phosphorylcholine group of PC. The quantitative analysiss of the C6 stretching vibration bands of the chloroform-d solution of PC showed that the latter hydrogen bonding mainly contributes to the solubility and that PC dissolves in chloroform to form a complex consisting of a few or more molecules of chloroform and one molecule of PC. We discussed in this report about the molecular organization of PC in chloroform solution.     

1H- and 13C-NMR studies of N-acetyl-L-alanine methylester and N-acetyl-L-alanine methylamide. I. Self-association

The ¹H- and ¹³C-NMR spectra of N-acetyl-L -alanine methylester and N-acetyl-L -alanine methylamide were measured to examine the modes of self-association of these molecules in solution. The different dilution shifts between these molecules seem to correspond to the difference in the associated state for each molecule. Consequently, for the former molecule, a dimer model forming the intermolecular hydrogen bond through Ala NH hydrogen atom in one molecule to Ala C?O oxygen atom in another molecule was proposed. Another dimer model, which coincides with that proposed recently by Neel and coworkers, was proposed for the latter molecule. This second dimer model forms an intermolecular hydrogen bond through the NH of the N-methylamide group in one molecule to the acetyl C?O in another molecule.     

Conformation of histidine model peptides. I. Conformational energy calculations for L-alanyl-L-histidine diketopiperazine

Semiempirical conformational energy calculations were carried out for the cyclic dipeptide L -alanyl-L -histidine diketopiperazine. The results indicate that electrostatic effects are probably significant in determining the conformation assumed by this molecule. When the imidazole group is in its uncharged state the most stable conformations of the molecule are those with the imidazole ring folded over the diketopiperazine ring (χ¹ = 60°). Upon protonation of the imidazole group the folded conformation may be destabilized relative to conformations characterized by χ¹ positions near 180°.     

Infrared spectroscopy of the water vapor sorption process of caseins.

Infrared spectra of as-, beta- and micellar casein were studied at relative water vapor pressures (p/po) ranging from 0 to 0.98. The samples were prepared as self-supporting films by evaporating concentrated aqueous suspensions of the caseins under study. An infrared cell and a vacuum apparatus were constructed which allowed exposure of the casein films either to vacuum or to sorbate vapor. Following the increase in intensities of the OH and O2H absorption bands during hydration, a sigmoid-shaped curve was observed, similar to the type II isotherm usually obtained by gravimetric sorption measurements. The pronounced frequency and intensity changes in the amide I, II and III bands in the p/po range from 0 to about 0.10 lead to the conclusion that water molecules are already attached to the peptide repeat unit at very low humidities. Based on calculations of the amount of polar groups per casein molecule it was shown that much less than one water molecule per polar group is needed to cause these significant spectral changes.     

On the catalytic mechanism of EcoRI and EcoRV. A detailed proposal based on biochemical results, structural data and molecular modelling.

EcoRI and EcoRV have a very similar active site, as is apparent from a comparison of the structures of their respective protein-DNA complexes. Based on structural and mechanistic data, as well as detailed molecular modelling presented here, a mechanism for the DNA cleavage by these enzymes is suggested in which the attacking water molecule is activated by the phosphate group 3' to the scissile phosphodiester bond, and in which the leaving group is protonated by a water molecule associated with the essential cofactor, Mg2+. The mechanism proposed may also apply to other nucleases.     

Isolation of a prokaryotic plasmin receptor. Relationship to a plasminogen activator produced by the same micro-organism.

Plasminogen receptors have been identified on the surface of a number of prokaryotic and eukaryotic cells. A receptor demonstrating high affinity for plasmin with minimal reactivity with the native zymogen Glu-plasminogen has been identified on the surface of certain group A streptococci. In this study the group A streptococcal plasmin receptor has been solubilized and purified to homogeneity. The isolated protein was an Mr approximately 41,000 molecule which retained its ability to bind plasmin following solubilization and affinity purification on a column of enzymatically inactivated human plasmin. The isolated plasmin receptor was compared functionally, antigenically, and physicochemically to the secreted plasminogen activator, streptokinase, produced by the same organism. The Mr approximately 41,000 surface plasmin receptor was shown to be functionally and antigenically distinct from the Mr approximately 48,000 streptokinase molecule produced by the same strain and lacked any plasminogen activator activity. The streptokinase molecule produced by this strain was shown to be closely related to the plasminogen activator protein secreted by other group A and C streptococci. This study represents the first report of the isolation of a plasmin receptor, either prokaryotic or eukaryotic, with functional activity.