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Glycosaminoglycans (GAGs) have been suggested to be a potential target for cobra cardiotoxin (CTX) with high affinity and specificity via a cationic belt at the concave surface of the polypeptide. The interaction of GAGs, such as high-molecular weight heparin, with CTXs not only can induce aggregation of CTX molecules but also can enhance their penetration into membranes. The binding of short chain heparin, such as a heparin-derived disaccharide [DeltaUA2S(1-->4)-alpha-D-GlcNS6S], to CTX A3 from Taiwan cobra (Naja atra), however, will not induce aggregation and was, therefore, investigated by high-resolution (1)H NMR. A novel heparin binding site on the convex side of the CTX, near the rigid disulfide bond-tightened core region of Cys38, was identified due to the observation of intermolecular NOEs between the protein and carbohydrate. The derived carbohydrate conformation using complete relaxation and conformational exchange matrix analysis (CORCEMA) of NOEs indicated that the glycosidic linkage conformation and the ring conformation of the unsaturated uronic acid in the bound state depended significantly on the charge context of CTX molecules near the binding site. Specifically, comparative binding studies of several heparin disaccharide homologues with two CTX homologues (CTX Tgamma from Naja nigricollis and CTX A3) indicated that the electrostatic interaction of N-sulfate of glucosamine with NH(3)(+)zeta of Lys12 and of the 2-O-sulfate of the unsaturated uronic acid with NH(3)(+)zeta of Lys5 played an important role. These results also suggest a model on how the CTX-heparin interaction may regulate heparin-induced aggregation of the toxin via the second heparin binding site.

Chronic parasitic infections are a major risk factor for cancer development in many underdeveloped countries. Oxidative damage of DNA may provide a mechanism linking these processes. Eosinophil recruitment is a hallmark of parasitic infections and many forms of cancer, and eosinophil peroxidase (EPO), a secreted hemoprotein, plays a central role in oxidant production by these cells. However, mechanisms through which EPO may facilitate DNA oxidation have not been fully characterized. Here, we show that EPO effectively uses plasma levels of bromide as a cosubstrate to brominate bases in nucleotides and double-stranded DNA, forming several stable novel brominated adducts. Products were characterized by HPLC with on-line UV spectroscopy and electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). Ring assignments for brominated purine bases as their 8-bromo adducts were identified by NMR spectroscopy. Using stable isotope dilution LC/ESI/MS/MS, we show that while guanine is the preferred purine targeted for bromination as a free nucleobase, 8-bromoadenine is the major purine oxidation product generated following exposure of double-stranded DNA to either HOBr or the EPO/H(2)O(2)/Br(-) system. Bromination of nucleobases was inhibited by scavengers of hypohalous acids such as the thioether methionine, but not by a large molar excess of primary amines. Subsequently, N-monobromoamines were demonstrated to be effective brominating agents for both free nucleobases and adenine within intact DNA. A rationale for selective modification of adenine, but not guanine, in double-stranded DNA based upon stereochemical criteria is presented. Collectively, these results suggest that specific brominated DNA bases may serve as novel markers for monitoring oxidative damage of DNA and the nucleotide pool by brominating oxidants.

An analogue of lysine, trans-4,5-dehydro-L-lysine (trans-4, 5-dehydrolysine), is a potent inhibitor of lysine 2,3-aminomutase from Clostridium subterminale SB4 that competes with L-lysine for binding to the active site. Inclusion of trans-4,5-dehydrolysine with activated enzyme and the coenzymes pyridoxal-5'-phosphate and S-adenosylmethionine, followed by freezing at 77 K, produces an intense signal in the electron paramagnetic resonance (EPR) spectrum at g 2.0, which is characteristic of an organic radical. A series of deuterated and (15)N-labeled samples of trans-4,5-dehydrolysine were synthesized and used to generate the EPR signal. Substitution of deuterium for hydrogen at C2, C3, C4, C5, and C6 of trans-4, 5-dehydrolysine led to significant simplifications and narrowing of the EPR signal, showing that the unpaired electron was located on the carbon skeleton of 4,5-trans-4,5-dehydrolysine. The hyperfine splitting pattern is simplified by use of 4,5-dehydro[3, 3-(2)H(2)]lysine or 4,5-dehydro[4,5-(2)H(2)]lysine, and it is dramatically simplified with 4,5-dehydro-[3,3,4,5,6,6-(2)H(6)]lysine. Spectral simulations show that the EPR signal arises from the allylic radical resulting from the abstraction of a hydrogen atom from C3 of trans-4,5-dehydrolysine. This radical is an allylic analogue of the substrate-related radical in the rearrangement mechanism postulated for this enzyme. The rate constant for formation of the 4,5-dehydrolysyl radical (2 min(-)(1)) matches that for the decrease in the concentration of [4Fe-4S](+), showing that the two processes are coupled. The cleavage of S-adenosylmethionine to 5'-deoxyadenosine and methionine takes place with a rate constant of approximately 5 min(-)(1). These kinetic correlations support the hypothesis that radical formation results from a reversible reaction between [4Fe-4S](+) and S-adenosylmethionine at the active site to form [4Fe-4S](2+), the 5'-deoxyadenosyl radical, and methionine as intermediates.

One molecule of MgATP binds to each subunit of the homodimeric Fe protein component of nitrogenase. Both MgATP molecules are hydrolyzed to MgADP and P(i) in reactions coupled to the transfer of one electron into the MoFe protein component. As an approach to assess the contributions of individual ATP binding sites, a heterodimeric Fe protein was produced that has an Asn substituted for residue 39 in the ATP binding domain in one subunit, while the normal Asp(39) residue within the other subunit remains unchanged. Separation of the heterodimeric Fe protein from a mixed population with homodimeric Fe proteins contained in crude extracts was accomplished by construction of a seven His tag on one subunit and a differential immobilized-metal-affinity chromatography technique. Three forms of the Fe protein (wild-type homodimeric Fe protein [Asp(39)/Asp(39)], altered homodimeric Fe protein [Asn(39)/Asn(39)], and heterodimeric Fe protein [Asp(39)/Asn(39)]) were compared on the basis of the biochemical and biophysical changes elicited by nucleotide binding. Among those features examined were the MgATP- and MgADP-induced protein conformational changes that are manifested by the susceptibility of the [4Fe-4S] cluster to chelation and by alterations in the electron paramagnetic resonance, circular dichroism, and midpoint potential of the [4Fe-4S] cluster. The results indicate that changes in the [4Fe-4S] cluster caused by nucleotide binding are the result of additive conformational changes contributed by the individual subunits. The [Asp(39)/Asn(39)] Fe protein did not support substrate reduction activity but did hydrolyze MgATP and showed MgATP-dependent primary electron transfer to the MoFe protein. These results support a model where each MgATP site contributes to the rate acceleration of primary electron transfer, but both MgATP sites must be functioning properly for substrate reduction. Like the altered homodimeric [Asn(39)/Asn(39)] Fe protein, the heterodimeric [Asp(39)/Asn(39)] Fe protein was found to form a high affinity complex with the MoFe protein, revealing that alteration on one subunit is sufficient to create a tight complex.

A variety of chronic inflammatory conditions are associated with an increased risk for the development of cancer. Because of the numerous links between DNA oxidative damage and carcinogenesis, a potential role for leukocyte-generated oxidants in these processes has been suggested. In the present study, we demonstrate a novel free transition metal ion-independent mechanism for hydroxyl radical ((*)OH)-mediated damage of cellular DNA, RNA, and cytosolic nucleotides by activated neutrophils and eosinophils. The mechanism involves reaction of peroxidase-generated hypohalous acid (HOCl or HOBr) with intracellular superoxide (O(2)(*)(-)) forming (*)OH, a reactive oxidant species implicated in carcinogenesis. Incubation of DNA with either isolated myeloperoxidase (MPO) or eosinophil peroxidase (EPO), plasma levels of halides (Cl(-) and Br(-)), and a cell-free O(2)(*)(-) -generating system resulted in DNA oxidative damage. Formation of 8-hydroxyguanine (8-OHG), a mutagenic base which is a marker for (*)OH-mediated DNA damage, required peroxidase and halides and occurred in the presence of transition metal chelators (DTPA +/- desferrioxamine), and was inhibited by catalase, superoxide dismutase (SOD), and scavengers of hypohalous acids. Similarly, exposure of DNA to either neutrophils or eosinophils activated in media containing metal ion chelators resulted in 8-OHG formation through a pathway that was blocked by peroxidase inhibitors, hypohalous acid scavengers, and catalytically active (but not heat-inactivated) catalase and SOD. Formation of 8-OHG in target cells (HA1 fibroblasts) occurred in all guanyl nucleotide-containing pools examined following exposure to both a low continuous flux of HOCl (at sublethal doses, as assessed by [(14)C]adenine release and clonogenic survival), and hyperoxia (to enhance intracellular O(2)(*)(-) levels). Mitochondrial DNA, poly A RNA, and the cytosolic nucleotide pool were the primary targets for oxidation. Moreover, modest but statistically significant increases in the 8-OHG content of nuclear DNA were also noted. These results suggest that the peroxidase-H(2)O(2)-halide system of leukocytes is a potential mechanism contributing to the well-established link between chronic inflammation, DNA damage, and cancer development.

The mechanism of orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) was studied using the decarboxylation of orotic acid analogues as a model system. The rate of decarboxylation of 1,3-dimethylorotic acid and its analogues as well as the stability of their corresponding carbanion intermediates was determined. The results have shown that the stability of the carbanion intermediate is not a critical factor in the rate of decarboxylation. On the other hand, the reaction rate is largely dependent on the equilibrium constant for the formation of a zwitterion. Based on these results, we have proposed a new mechanism in which ODCase catalyzes the decarboxylation of OMP by binding the substrate in a zwitterionic form and providing a destabilizing environment for the carboxylate group of OMP.

The initiation of lipid peroxidation and the concomitant formation of biologically active oxidized lipids and sterols is believed to play a central role in the pathogenesis of inflammatory and vascular disorders. Here we explore the role of neutrophil- and myeloperoxidase (MPO)-generated nitrating intermediates as a physiological catalyst for the initiation of lipid peroxidation and the formation of biologically active oxidized lipids and sterols. Activation of human neutrophils in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide (nitrogen monoxide, NO) metabolism, generated an oxidant capable of initiating peroxidation of lipids. Formation of hydroxy- and hydroperoxyoctadecadienoic acids [H(P)ODEs], hydroxy- and hydroperoxyeicosatetraenoic acids [H(P)ETEs], F(2)-isoprostanes, and a variety of oxysterols was confirmed using on-line reverse phase HPLC tandem mass spectrometry (LC/MS/MS). Lipid oxidation by neutrophils required cell activation and NO(2)(-), occurred in the presence of metal chelators and superoxide dismutase, and was inhibited by catalase, heme poisons, and free radical scavengers. LC/MS/MS studies demonstrated formation of additional biologically active lipid and sterol oxidation products known to be enriched in vascular lesions, such as 1-hexadecanoyl-2-oxovalaryl-sn-glycero-3-phosphocholine, which induces upregulation of endothelial cell adhesion and chemoattractant proteins, and 5-cholesten-3beta-ol 7beta-hydroperoxide, a potent cytotoxic oxysterol. In contrast to the oxidant formed during free metal ion-catalyzed reactions, the oxidant formed during MPO-catalyzed oxidation of NO(2)(-) readily promoted lipid peroxidation in the presence of serum constituents. Collectively, these results suggest that phagocytes may employ MPO-generated reactive nitrogen intermediates as a physiological pathway for initiating lipid peroxidation and forming biologically active lipid and sterol oxidation products in vivo.

Detection of specific reaction products is a powerful approach for dissecting out pathways that mediate oxidative damage in vivo. Eosinophil peroxidase (EPO), an abundant protein secreted from activated eosinophils, has been implicated in promoting oxidative tissue injury in conditions such as asthma, allergic inflammatory disorders, cancer, and helminthic infections. This unique heme protein amplifies the oxidizing potential of H2O2 by utilizing plasma levels of Br- as a cosubstrate to form potent brominating agents. Brominated products might thus serve as powerful tools for identifying sites of eosinophil-mediated tissue injury in vivo; however, structural identification and characterization of specific brominated products formed during EPO-catalyzed oxidation have not yet been reported. Here we explore the role of EPO and myeloperoxidase (MPO), a related leukocyte protein, in promoting protein oxidative damage through bromination and demonstrate that protein tyrosine residues serve as endogenous traps of reactive brominating species forming stable ring-brominated adducts. Exposure of the amino acid L-tyrosine to EPO, H2O2, and physiological concentrations of halides (100 mM Cl-,

We have reported that some cardiotoxins (CTXs), homologous basic polypeptides of cobra venom, bind strongly to heparin. Herein we show that CTXs from spitting cobra venom bind avidly to chondroitin-6-sulfate (CS6) and dermatan sulfate (DS), the glycosaminoglycans (GAGs) abounding in the cornea and elsewhere. We compared the binding strength of Tgamma, a major component of spitting cobra, Naja nigricollis, venom with that of CTX A3, a major component of Naja atra venom to various GAGs including CS6, chondroitin-4-sulfate (CS4), DS, keratan sulfate (KS), hyaluronan (HYA), and heparin. The binding strength of Tgamma followed the order CS6 > KS > HYA > DS > CS4 > heparin, whereas that of CTX A3 was heparin > KS > CS4 > DS > CS6 > HYA. The binding specificity displayed by different CTXs toward GAGs is impressive, given the high homology among CTXs and among GAGs. Strong binding of Tgamma to CS6, rather than to the highly anionic and versatile cousin, heparin, implies specific interaction with CS6. Heparin, at high concentration, displaced CS6 from CS6-Tgamma and CS6-A3 complexes. We also show that corneal CS/DS likely allow Tgamma to bind to corneal epithelium. CTXs of spitting cobra venom are known to cause corneal opacity and/or blindness. Taken together with these observations, our results suggest that corneal CS/DS play a role in the action of CTX in the eye. Most importantly, the present results establish CTXs as cationic, readily available, avidly binding ligands of CS/DS.

The interaction of Taiwan cobra cardiotoxin (CTX A3), a basic polypeptide consisting of three-fingered loops and five-strand beta-sheet structure, with zwitterionic dipalmitoylphosphatidylcholine (DPPC) has been studied by 31P and 2H NMR to understand the binding modes of CTX in membrane bilayers. The results, in conjunction with DPH fluorescence anisotropy and differential scanning calorimetry studies, show that CTX may penetrate and lyse the bilayers into small aggregates at a lipid/protein molar ratio of about 20 in the ripple Pbeta' phase. Elevating the temperature to that of the liquid crystalline Lalpha phase leads to the fusion of the small aggregates into larger ones as evidenced by the change of the isotropic signal into a magnetically aligned 31P signal with a marked reduction in the chemical shift anisotropy. 2H NMR study on deuterium-labeled DPPC in the head group and fatty acyl region as a function of temperature and CTX concentration reveals a molecular model that CTX undergoes a redistribution between penetrating and peripheral binding states depending on the temperature studied. In addition, both the conformational and dynamic states of the phosphocholine head group of DPPC bilayers are significantly perturbed in the presence of CTX. Structural consideration of the CTX molecule indicates that the penetration binding mode of CTX with the DPPC bilayer may involve a novel membrane-binding motif identified recently in the three-fingered loops of P-type CTX. CTX can only bind to DPPC membrane peripherally in the Lalpha phase due to the mismatch of their hydrophobic lengths.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of lysine and beta-lysine by a mechanism in which four organic radicals are postulated as intermediates. One of the intermediates has been identified as the alpha-radical of beta-lysine in imine linkage to pyridoxal phosphate (PLP) [Ballinger, M. D., Frey, P. A., & Reed, G. H. (1992) Biochemistry 31, 10782-10788]. We report here the observation of another of the four putative radical intermediates in the reaction of the alternative substrate, 4-thia-L-lysine (S-2-aminoethyl-L-cysteine). 4-Thialysine is a substrate for lysine 2,3-aminomutase. The Km of 4-thialysine is similar to that for lysine, and the Vm is approximately 3% of that for lysine. Upon mixing 4-thialysine with the activated enzyme in the presence of the required cofactor S-adenosylmethionine, followed by freeze-quenching with liquid N2 in the steady state, a strong EPR signal centered at g = 2.003 is observed. This signal exhibits strong hyperfine splitting due to the presence of 13C at carbon-3 of 4-thialysine, and the EPR pattern is narrowed upon the substitution of deuterium at carbon-3. The hyperfine interactions show that the unpaired electron is centered on carbon-3 of 4-thialysine. The hyperfine pattern in the EPR spectrum is also simplified by the use of 4-thia[5,6-2H4]lysine as the substrate, showing either that the spin is partially delocalized through the sulfur intervening between carbons-3 and -5 or that the conformation is such that protons at carbon-6 are close to carbon-3.(ABSTRACT TRUNCATED AT 250 WORDS)

The RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-DNA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the last 19 amino acids, were expressed. In both cases, these RTs made multiple cuts in PPT-containing substrates, whereas wild-type cleavages occurred primarily at sites consistent with the distance between the polymerase and RNase H active sites. Primer extension assays performed with the chimeric RTs, an RNase H-minus RT, and wild-type showed that the presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wild-type RT was bound to primer-template, two retarded bands could be detected in band-shift assays. In the absence of primer extension, a high proportion of enzyme-bound primer-template was associated with the faster-migrating band, whereas with DNA synthesis, more of the bound radioactivity was in the super-shifted complex. This suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this complex, but the chimeric RTs were somewhat less defective than the RNase H-minus mutant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer-template.

The mechanism of iron-bleomycin-mediated ds-cleavage of DNA has been investigated at specific sites within specific sequences using hairpin oligonucleotides (Absalon et al., 1995) and our recently developed technique for determining sequence-specific isotope effects upon oxidative degradation of DNA (Kozarich et al., 1989; Worth et al., 1993). Isotope effects upon ds-cleavage have been observed when the C-4' hydrogen of either nucleotide involved in the ds-break was substituted with deuterium. The values of the isotope effects determined for ss and ds events occurring at the same site were indistinguishable at four sites examined in detail. The results are consistent with a mechanism of ds-cleavage in which the pathways leading to ss- and ds-cleavage partition from a common intermediate subsequent to abstraction of the C-4' hydrogen from the first nucleotide involved in the cleavage. Deuterium substitution at the primary cleavage site of a ds-break failed to result in an equivalent effect on the amount of cleavage at the secondary cleavage site, suggesting that ds-cleavage may be initiated from either of the nucleotides involved in the ds-cleavage event. A kinetic preference for cleavage initiated at the 1 degree site, however, is probable. The requirement in the ds-cleavage process for O2, in addition to that needed to form "activated BLM", has been clearly demonstrated by the absence of ds-cleavage products in reactions performed under anaerobic conditions in which ss-cleavage still occurs. These results support, in part, the basic model for ds-cleavage proposed by Steighner and Povirk [(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8350-8354], in which a single molecule of BLM effects ds-cleavage and requires reactivation to effect cleavage at the second strand. The essential factor establishing the ratio of ss- to ds-cleavage at a specific site may be related to the efficiency by which Fe-BLM can be reactivated and/or repositioned at a second site for cleavage.

Heme d1 is the noncovalently associated heme prosthetic group of the bacterial nitrite reductase known as cytochrome cd1. Additional evidence has been obtained in support of a dioxoisobacteriochlorin, or 1,3-porphyrindione, skeleton for this heme. The new data include the natural abundance 13C NMR spectrum of the free base methyl ester derivative of d1, mass spectrometric determinations of the molecular mass of the free base methyl ester and the Cu and the Zn chelates, visible and 1H NMR spectral comparisons between d1 and synthetic porphyrindione model compounds, and the isolation and characterization of several byproducts formed during the purification of the free base methyl ester of d1. The accumulated evidence strongly supports the following structure for the skeleton of d1: 1-oxo-2-methyl-2'-acetyl-3-oxo-4-methyl-4'-acetyl-5-methyl-6-acrylyl+ ++-7- propionyl-8-methylporphyrin.