Theoretical studies on protein-nucleic acid interactions. III. Stacking of aromatic amino acids with bases and base pairs of nucleic acids

Stacking of aromatic amino acids tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), and histidine (His) with bases and base pairs of nucleic acids has been studied. Stacking energies of the amino acid–base (or base pair) complexes have been calculated by second-order perturbation theory. Our results show that, in general, the predominant contribution to the total stacking energy comes from the dispersion terms. In these cases, repulsion energy is greater than the sum of electrostatic and polarization energies. In contrast to this, interaction of histidine with the bases and base pairs is largely Coulombic in nature. The complexes of guanine with aromatic amino acids are more stable than the corresponding complexes of adenine. Among pyrimidines, cytosine forms the most stable complexes with the aromatic amino acids. The G · C base pair has the highest affinity with aromatic amino acids among various sets of base pairs. Optimized geometries of the stacked complexes show that the aromatic moieties overlap only partially. The heteroatom of one residue generally overlaps with the other aromatic moiety. There is a considerable degree of configurational freedom in the stacked geometries. The role of stacking in specific recognition of base sequences by proteins is discussed.     

Nucleophilic addition reactions of free and enzyme-bound deazaflavin.

DeazaFMN-containing glycolate oxidase has been prepared and shown to catalyze the stereospecific transfer of the alpha-hydrogen from substrate to enzyme-bound deazaFMN. The reaction of sulfite, cyanide, and hydroxylamine with several deazaflavin-containing enzymes (glycolate oxidase, D-amino acid oxidase, glucose oxidase, N-methylglutamate synthetase) and free deazaFMN has been examined. All the deazaflavin systems tested form reversible 1:1 complexes with sulfite and cyanide. The pH dependence of the reaction of free deazaFMN with cyanide indicates that cyanide anion is the reacting nucleophile. Hydroxylamine complexes are formed with deazaFMN glycolate oxidase and deazaFAD glucose oxidase. The effectiveness of the various nucleophilic reagents in complex formation decreases in the following order: sulfite greater than cyanide greater than hydroxylamine. The relative stability observed for the sulfite and cyanide complexes formed with various deazaflavin systems (glycolate oxidase greater than D-amino acid oxidase greater than free deazaFMN) follows the same trend observed for the stability of the sulfite complexes formed with the corresponding flavin system. A correlation is also observed between the reduction potential (E'o) of the deazaflavin system (glycolate oxidase (- 170 mV) greater than D-amino acid oxidase (-240 mV) greater than free deazaFMN (-178 mV) and the stability of the deazaflavin-nucleophile complexes. The following evidence indicates that deazaflavin systems are generally more susceptible toward nucleophilic attack than corresponding flavin system: (a) with the exception of glucose oxidase, the dissociation constants for the deazaflavin-sulfite complexes are at least 1 order of magnitude less than the corresponding flavin sulfite complexes; (b) the least reactive nucleophile, hydroxylamine, does not form a complex with any of the flavin systems. In the case of cyanide, a complex is formed only with native glycolate oxidase, which is the flavin-containing system most susceptible to attack by the more reactive sulfite. Formation of the various (deaza)flavin-nucleophile complexes is characterized by a bleaching of the longer wavelength absorption band of the chromophore and increases in absorption below the isosbestic point of the reaction in the near-ultraviolet region of the spectrum. These results are consistent with the formation of covalent adducts via attack of the various nucleophiles at position 5 of (deaza)flavin. The reaction with cyanide provides the first example of a reversible addition of carbanion to enzyme-bound (deaza)flavin.     

Oligonucleotide interactions. I. Structure of 2:1 complexes between polyuridylic acid and oligoadenylic acid

The circular dichroism of a number of 1:2 complexes between oligoadenylic acid and polyuridylic acid has been measured. The structure of these complexes, as evidenced by their optical properties, is independent of the chain length of the oligonucleotide throughout all ranges of chain lengths from adenosine to high molecular weight polyadenylic acid. A similar structure was found for the complex (Ap)₅A:2(Up)₅U.     

N.m.r. study on the formation and geometry of inclusion complexes of 6-O-(alpha-maltosyl)cyclomalto-hexaose and -heptaose with p-nitrophenol in aqueous solution.

The formation and molecular geometry of inclusion complexes of some branched cyclomaltaoses with p-nitrophenol in aqueous solution have been investigated by using high-resolution 1H-n.m.r. spectroscopy. 6-O-(alpha-Maltosyl)cyclomalto-hexaose and -heptaose were found to form 1:1 inclusion complexes with p-nitrophenol, and the dissociation constants for their complexes are quite similar to those for corresponding unbranched cyclomaltaose-p-nitrophenol complexes, indicating that formation of these inclusion complexes is not hampered by the maltosyl branch. From measurement of nuclear Overhauser enhancements, it was concluded that the maltosyl branch is not situated over the entrance of the cavity.     

Water-soluble platinum(II) complexes of diamine chelating ligands bearing amino-acid type substituents: the effect of the linked amino acid and the diamine chelate ring size on antitumor activity, and interactions with 5'-GMP and DNA

Six new Pt(II) complexes are described having the general formula PtCl(2)(LL), in which LL is a chelating diamine ligand bearing an amino acid as substituent. The amino acids chosen are l-alanine and its methyl ester, and l-phenylalanine. The compounds have been characterized using analytical and spectroscopic methods. The influence on the biological properties of the size of the chelate ring and the structure of the amino acid substituent has been studied. The effect of the presence of a carboxylic or carboxylate group on the amino acid C-terminus has also been determined. It is demonstrated by circular dichroism (CD) that the effect on the secondary structure of DNA induced by the six complexes differ from each other. In all cases, the interaction takes place at the N7 position of the purine bases, as shown by NMR monitoring. The general behavior of these platinum complexes, with one exception, is to uncoil the DNA from the B form to the C form. The interactions with 5'-GMP and DNA have been compared with their expected antitumour activity. The complexes with l-alanine and l-phenylalanine exhibit cytotoxic activity in HeLa and HL-60 cell lines, in a dose- and time-dependent manner. No cytotoxic activity of the methyl ester derivatives have been determined because of their low solubility in aqueous solution.     

The interaction of trifluoroacetyl peptide chloromethyl ketones with porcine pancreatic elastase. Direct evidence for nonproductive enzyme.inhibitor complexes.

19F NMR investigations of the interactions between elastase and the reversible inhibitors CF3CO-Ala3, CF3CO-Lys-Ala2 and CF3CO-Ala4 show that these peptides have a single mode of binding to the enzyme. Furthermore the results indicate that the CF3CO-group experiences the same environment in all of the reversible complexes formed with these inhibitors. This agrees with the higher affinity of these peptides for the enzyme as compared to the corresponding acetylated inhibitors and confirms our earlier hypothesis of the existence of a specific binding site for the CF3CO-group on the enzyme. The interactions between elastase and the irreversible inhibitors CF3CO-Alan chloromethyl ketone (n = 2, 3, 4, 5) and CF3CO-Lys-Ala4 chloromethyl ketone have been investigated by enzymatic measurements and 19F NMR spectroscopy. The kinetic constants k2 and KI describing the irreversible inhibition are significantly lower for all the CF3CO-peptide chloromethyl ketones with exception of CF3CO-Ala2 chloromethyl ketone, than for the corresponding acetylated ones. Moreover, 19F NMR spectroscopy enabled us to demonstrate, for the tri- and tetrapeptide derivatives, the parallel formation of reversible nonproductive enzyme.inhibitor complexes. The spectroscopic properties of these complexes are completely different from those of the irreversible complexes but are similar to those observed with the reversible complexes described above. In the case of the pentapeptide chloromethyl ketones, fast hydrolysis of the peptide or fast inactivation of the enzyme does not allow observations to be made but does not exclude the existence of reversible nonproductive complexes. In fact, their existence is strongly suggested by the enzyme reaction rate measurements. The similarity of the properties of all the reversible complexes, their striking differences with those of all the irreversible complexes, as well as their mutual exclusivity, permit the conclusion that the CF3CO-group does not bind at one of the classical S subsites of elastase.     

Identification of specific residues involved in substrate discrimination in two plant O-methyltransferases.

Among the large number of plant O-methyltransferases that are involved in secondary metabolism, only a few have been enzymatically characterized, and little information is available on the structure of their substrate binding site and the mechanism which determines their substrate specificity and methylation regiospecificity. We have previously reported the isolation of two O-methyltransferases, S-adenosyl-l-methionine:(iso)eugenol O-methyltransferase (IEMT) and S-adenosyl-l-methionine:caffeic acid O-methyltransferase (COMT) from Clarkia breweri, an annual plant from California. While IEMT and COMT (which methylate eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid, respectively) share 83% identity at the amino acid level, they have distinct substrate specificity and methylation regiospecificity. We report here that seven amino acids play a critical role in discriminating between eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid. When these amino acids in IEMT were replaced by the corresponding residues of COMT, the hybrid protein showed activity only with caffeic acid/5-hydroxyferulic acid. Conversely, when these amino acids in COMT were replaced by corresponding IEMT residues, the hybrid protein had activity only with eugenol/isoeugenol. These results provide strong evidence that O-methyltransferase substrate preference could be determined by a few amino acid residues and that new OMTs with different substrate specificity could begin to evolve from an existing OMT by mutation of a few amino acids. Phylogenetic analysis confirms that C. breweri IEMT evolved recently from COMT.     

Plasmid-protein relaxation complexes in Staphylococcus aureus.

Protein-deoxyribonucleic acid relaxation complexes have been demonstrated for six Staphylococcus aureus plasmids out of sixteen examined. Four of these encode stretomycin resistence, have molecular weights of about 2.7 x 10(6), and are isolated as supercoiled molecules that are virtally 100% relaxable by treatment with sodium dodecyl sulfate. It is probable that these four isolates represent a single widely disseminated plasmid species. The other two plasmids showing relaxation complexes have molecular weights of about 3 x 10(6) and encode chloramphenicol resistance. The complexes in these cases are unstable, and it has not been possible to induce more than 50% relaxation by any of the standard treatments. Ten other plasmids do not show detectable complexes. These include three penicillinase plasmids, four tetracycline-resistance plasmids, one plasmid carrying kanamycin-neomycin resistance, and finally, two chloramphenicol-resistance plasmids.     

Nickel-quinolones interaction. Part 4. Structure and biological evaluation of nickel(II)-enrofloxacin complexes compared to zinc(II) analogues

The nickel(II) complexes with the second-generation quinolone antibacterial agent enrofloxacin in the presence or absence of the nitrogen-donor heterocyclic ligands 1,10-phenanthroline, 2,2′-bipyridine or pyridine have been synthesized and characterized. Enrofloxacin acts as bidentate ligand coordinated to Ni(II) ion through the ketone oxygen and a carboxylato oxygen. The crystal structure of (1,10-phenanthroline)bis(enrofloxacinato)nickel(II) has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and bis(pyridine)bis(enrofloxacinato)nickel(II) exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the complexes have been evaluated in comparison to the corresponding Zn(II) enrofloxacinato complexes as well as Ni(II) complexes with the first-generation quinolone oxolinic acid.     

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence of C9 consists of 261 amino acid residues with a calculated molecular weight of 29,496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.     

Synthesis and properties of poly 5-methylthiouridylic acid.

In an effort to search for good methods for the enzymatic synthesis of polynucleotide analogs with antitemplate activity, 5-methylthiouridine-5'-diphosphate (ms5UDP) has been synthesized and investigated as a substrate for polynucleotide phosphorylase. While ms5UDP was polymerized at a very low rate to give a 6% yield of polynucleotides by the polynucleotide phosphorylase of Micrococcus luteus, it was utilized more efficiently by the corresponding enzyme of Escherichia coli resulting in a 15% yield of poly (5-methylthiouridylic) acid. Results of the co-polymerization of ms5UDP and UDP revealed that the ratio of 5-methylthiouridylate to uridylate residues in the polynucleotide product was lower than the ratio of ms5UDP to UDP in the substrate mixture. The 5-methylthio group conferred only minute changes on the conformation of the modified polyuridylic acid, and the complexes formed between poly-(5-methylthiouridylic) acid and poly(adenylic) acid possessed slightly higher Tm values than did the unmodified counterparts. Poly(5-methylthiouridylic) acid was a potent inhibitor of calf thymus DNA polymerase alpha.     

Some potential anticancer palladium(II) complexes of 2,2'-bipyridine and amino acids

Nine new palladium(II) complexes of the formula [Pd(bipy)(AA)]n+ (where bipy is 2,2'-bipyridine, AA is an anion of L-cysteine, L-aspartic acid, L-glutamic acid, L-methionine, L-histidine, L-arginine, L-phenylalanine, L-tyrosine, or L-tryptophan, and n = 0 or 1) have been synthesized by interaction of [Pd(bipy)Cl2] with an appropriate sodium salt of amino acid in water. These palladium(II) complexes have been characterized by chemical analysis and by visible, infrared, and 1H NMR spectroscopy. The modes of binding of amino acids in these palladium complexes have been ascertained by infrared and 1H NMR spectroscopy. The molar conductances of these complexes in water suggest that they are either nonelectrolytes or 1:1 electrolytes. These palladium complexes have shown growth inhibition against L1210 lymphoid leukemic, P388 lymphocytic leukemic, Sarcama 180, and Ehrlich ascites tumor cells. Some of these complexes show I.D.50 values comparable to or lower than cis-diamminedichloroplatinum(II).     

Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands

The complexation of glutathione and related ligands by the nitrilotriacetic acid complex of Cd2+ (Cd(NTA)-) has been investigated by 1H NMR as a model for the coordination chemistry of Cd2+ and GSH in biological systems. Related ligands included glycine, glutamic acid, cysteine, N-acetylcysteine, penicillamine, N-acetylpenicillamine, mercaptosuccinic acid, and the S-methyl derivative of glutathione. The nature of the complexes formed was deduced from 1H NMR spectra of Cd(NTA)- and the ligands. Mixed ligand complexes (Cd(NTA)L) and single ligand complexes (CdLx) are formed with the thiol ligands, whereas only mixed ligand complexes form with glycine, glutamic acid and S-methylglutathione. Formation constants of the mixed and the single ligand complexes were determined from NMR data. The results indicate that formation constants for binding of a thiolate donor group by Cd2+, either as the free ion or in a coordinately unsaturated complex, are in the range 10(5)-10(6).     

Magnesium-sugar interaction. Synthesis, spectroscopic and structural characterization of Mg-sugar complexes containing beta-D-fructose

The interaction between beta-D-fructose and hydrated magnesium salts has been studied and complexes of the type Mg(beta-D-fructose)Cl2.4H2O and Mg(beta-D-fructose)Br2.4H2O have been isolated and characterized. On the basis of comparisons of the spectroscopic and other chemical properties of several structurally known calcium-fructose compounds with those of the corresponding magnesium complexes, it is concluded that Mg2+ binds to two sugar moieties via O(2), O(3) of the first and O(4), O(5) of the second and to two water molecules, resulting in a six-coordinate geometry around the Mg2+. The strong sugar hydrogen-bonding network is rearranged upon sugar metallation and the sugar moiety shows the beta-anomer conformation in these magnesium-sugar complexes.     

Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA. IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA

Specimen-tilting in an electron microscope was used to determine the three-dimensional architecture of the helical complexes formed with DNA by the closely related single-stranded DNA binding proteins of fd and IKe filamentous viruses. The fd gene 5 protein is the only member of the DNA-helix-destabilizing class of proteins whose structure has been determined crystallographically, and yet a parameter essential to molecular modeling of the co-operative interaction of this protein with DNA, the helix handedness, has not been available prior to this work. We find that complexes formed by titrating fd viral DNA with either the fd or IKe gene 5 protein have a left-handed helical sense. Complexes isolated from Escherichia coli infected by fd virus are also found to be left-handed helical; hence, the left-handed fd helices are not an artefact of reconstitution in vitro. Because the proteins and nucleic acid of the complexes are composed of asymmetric units which cannot be fitted equivalently to right-handed and left-handed helices, these results rule out a previous computer graphics atomic model for the helical fd complexes: a right-handed helix had been assumed for the model. Our work provides a defined three-dimensional structural framework within which to model the protein-DNA and protein-protein interactions of two structurally related proteins that bind contiguously and co-operatively on single-stranded DNAs.     

Electronic substituent effects upon properties of 5-substituted-2-formylpyridine thiosemicarbazones and their metal complexes

The protonation constants of several 5-substituted-2-formylpyridine thiosemicarbazones, formation constants for their copper complexes, adduct formation constants of these complexes with ethylenediamine, protonation constants of the copper complexes, and half-wave reduction potentials of the copper and corresponding iron complexes have been determined. The electronic effect of substituents has been examined through the calculation of linear free energy correlations utilizing Hammet substituent constants as the independent parameter in the relationships. The effect of substituents upon the pharmacological properties of thiosemicarbazones is reconsidered here. The current results are used to suggest new experiments involving the reaction of 5-substituted-2-formylpyridine thiosemicarbazonato copper(II) complexes with Ehrlich cells.     

Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes.

The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-GlcNAc, GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-N-acetyl-D-xylosamine, and GlcNAc-beta(1 leads to 4-GlcNAc-beta(1 leads to 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-D-glucosamine, or N-acetyl-D-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-D-glycosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformation of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-beta(1 leads to 4)-MurNAc-beta(1leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is "looser" than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.     

Oligodeoxynucleotides covalently linked to intercalating dyes as base sequence-specific ligands. Influence of dye attachment site.

New molecules with high and specific affinity for nucleic acid base sequences have been synthesized. They involve an oligodeoxynucleotide covalently attached to an intercalating dye. Visible absorption spectroscopy and fluorescence have been used to investigate the binding of poly(rA) to octadeoxythymidylates substituted by a 9-aminoacridine derivative in different positions along the oligonucleotide chain. The 9-amino group of the acridine dye was linked through a polymethylene bridge to the 3''-phosphate, the 5''-phosphate, the fourth internucleotidic phosphate or to both the 3''- and 5''-phosphates. Different interactions of the acridine dye were exhibited by these different substituted oligodeoxynucleotides when they bind to poly(rA). The interaction was shown to be specific for adenine-containing polynucleotides. The stability of these complexes was compared with that of oligodeoxynucleotides substituted by an alkyl group on the 3''-phosphate. The increase in stability due to the presence of the intercalating dye has been determined from the comparison of melting temperatures. These results are discussed with respect to the strategy of synthesis of a new class of molecules with high affinity and high specificity for nucleic acid base sequences.