Modification of the active site of alkaline phosphatase by site-directed mutagenesis.

The catalytically essential amino acid in the active site of bacterial alkaline phosphatase (Ser-102) has been replaced with a cysteine by site-directed mutagenesis. The resulting thiol enzyme catalyzes the hydrolysis of a variety of phosphate monoesters. The rate-determining step of hydrolysis, however, is no longer the same for catalysis when the active protein nucleophile is changed from the hydroxyl of serine to the thiol of cysteine. Unlike the steady-state kinetics of native alkaline phosphatase, those of the mutant show sensitivity to the leaving group of the phosphate ester.

Natural antisense RNA/target RNA interactions: possible models for antisense oligonucleotide drug design.

Current antisense oligonucleotides designed for drug therapy rely on Watson-Crick base pairing for the specificity of interactions between antisense and target molecules. However, thermodynamically stable duplexes containing non-Watson-Crick pairs have been formed with synthetic oligonucleotides. There are also numerous examples of non-canonical base pairs that participate in stable intra- and inter-molecular RNA/RNA pairing in prokaryotic and eukaryotic cells. Several natural antisense RNA/target RNA duplexes contain looped-out and bulged positions as well as non-canonical pairs as exemplified by formation of the Escherichia coli antisense micF RNA/ompF mRNA duplex. Secondary structures and the phylogenetic conservation of nucleotide sequences are well characterized in this system. Natural antisense/ target interactions may serve as models for determining possible and optimal antisense/target interactions in oligonucleotide drug design.

DNA modification promoted by water-soluble nickel(II) salen complexes: a switch to DNA alkylation.

Reaction of a 17-base hairpin-forming oligonucleotide with [N,N'-bis(salicylaldehyde)-meso-1,2-bis(4- trimethylaminophenyl)ethylenediimino]nickel(II) perchlorate, 2, and KHSO5 produced two types of high molecular weight products, an alkaline-labile species and a nonalkaline-labile species, which co-migrated on gel electrophoresis. Upon treatment with piperidine, the base-labile derivative led to strand scission products only at accessible guanine residues that were not part of a Watson-Crick duplex. The formation of higher molecular weight species is proposed to occur via a highly reactive ligand-centered radical acting as a DNA alkylating agent.

Chemical reagents for investigating the major groove of DNA.

Chemical modification provides an inexpensive and rapid method for characterizing the structure of DNA and its association with drugs and proteins. Numerous conformation-specific probes are available, but most investigations rely on only the most common and readily available of these. The major groove of DNA is typically characterized by reaction with dimethyl sulfate, diethyl pyrocarbonate, potassium permanganate, osmium tetroxide, and, quite recently, bromide with monoperoxysulfate. This commentary discusses the specificity of these reagents and their applications in protection, interference, and missing contact experiments.

Role of oxygen during horseradish peroxidase turnover and inactivation.

Horseradish peroxidase catalyzed oxidation of phenol has been reinvestigated to determine the requirements of facile enzyme autoinactivation. Turnover of this peroxidase was monitored spectrophotometrically at 400 nm and found dependent on the concentration of phenol and hydrogen peroxide. The inactivation of the peroxidase required both substrates, phenol and H2O2, but surprisingly was also potentiated by molecular oxygen. Exclusion of diffusible superoxide or hydroxyl radicals had slight effect on product formation or loss of catalytic activity. A mechanism is proposed to explain the unanticipated role of oxygen during enzyme inactivation.

Facile interconversion of duplex structures formed by copolymers of d(CG).

Correlations between DNA sequence and reactivity have often been drawn with an implicit or explicit connection to duplex structure. An in vitro model using oligonucleotides of defined sequences has been developed to characterize a potential source of the hypersensitivity that naturally occurring regions of redundant sequence exhibit with many nucleases. S-1 nuclease was used here to diagnose the unusual hybridization of copolymeric DNA, d(CG)6, and related oligomers, through product and kinetic analysis. Fully complementary but redundant sequences reacted with this enzyme almost an order of magnitude faster than did heterogeneous fragments of DNA. Hydrolysis products of the copolymers indicated that conformations with unpaired termini were the sole substrates under these studies, and only a facile equilibrium between aligned and extended structures was required to explain the heightened reactivity of this DNA. For example, d(CG)6 was converted to d(CG)5 and d(CG)4 whereas d(CG)4C was initially processed to an octamer and then only later to a hexamer. Catalysis by S-1 exhibited no other substrate or product specificity; even the disordered bases in the loop region of a hairpin structure, d(CG)3T4(CG)3, did not provide sites of enhanced enzyme action. The rate of DNA consumption under standard conditions was proportional to the expected concentration of overhanging sequences rather than the absolute amount of DNA present. All initial attempts to saturate enzyme activity failed, and therefore, the rate of substrate formation through strand slippage was always faster than the catalytic depletion of unpaired bases. Only a low-energy transition state(s) must then separate the various hybridized species since this structural equilibration proceeded readily under conditions of 10 mM potassium phosphate, pH 7, 100 mM NaCl, and 22 degrees C.

The ensemble reactions of hydroxyl radical exhibit no specificity for primary or secondary structure of DNA.

Hydroxyl radical reacts at numerous sites within nucleic acids to form a wide range of derivatives yet the conformational specificity of only one of these processes, direct strand fragmentation, has received much attention to date. Since the deleterious effects of this radical are not likely limited to strand fragmentation in vivo, this report examined the conformational specificity expressed in a more general manner. For this, modification of DNA was induced by the hydroxyl radical generating system of H2O2 and Fe-EDTA. The ensemble rate of oxidation (nucleobase + deoxyribose backbone) was determined from the overall consumption of a series of oligonucleotides that were designed to model random coils and double helixes containing complementary and noncomplementary base pairing. The resulting pseudo-first order rate constants derived from this model system were relatively unaffected by nucleotide sequence or secondary structure and varied from only 0.022 to 0.048 s-1. Consequently, the indiscriminant nature of hydroxyl radical appears to persist beyond strand fragmentation to include nucleobase oxidation as well.

The use of 6-(difluoromethyl)indole to study the activation of indole by tryptophan synthase.

6-(Difluoromethyl)indole has been characterized and developed as a probe for the turnover of indole by the bifunctional enzyme, tryptophan synthase (alpha 2 beta 2). The neutral form of the indolyl species undergoes a slow and spontaneous hydrolysis to produce 6-formylindole with a rate constant (k1) of 0.0089 +/- 0.0001 min-1. The overall rate is independent of pH in the range of 3.5-10.5. Above pH 10.5, the observed rate increases are due to the high reactivity of the anionic form of the indole; deprotonation at N-1 accelerates hydrolysis by 10(4)-fold (k2, 97 +/- 2 min-1). The magnitude of this effect provides a technique for detecting the formation or stabilization of the anionic form of indole. 6-(Difluoromethyl)indole is recognized and processed by the beta subunit of tryptophan synthase. Selective inactivation of the beta subunit prevents enzymatic processing of 6-(difluoromethyl)indole. Chromatographic isolation and mass spectral analysis has identified 6-(difluoromethyl)tryptophan as the sole turnover product of the indolyl substrate. The lack of enzyme-promoted dehalogenation does not exclude the formation of an indole anion during turnover but rather the data suggest that rapid carbon-carbon bond formation (greater than 5300 min-1) prevents the accumulation of this anion.

6-(Difluoromethyl)tryptophan as a probe for substrate activation during the catalysis of tryptophanase.

A substrate analogue, 6-(difluoromethyl)tryptophan, was developed and characterized for mechanistic investigation of tryptophanase. The utility of this derivative was based on its ability to partition between fluoride elimination and carbon-carbon bond scission during tryptophan metabolism. The non-enzymatic hydrolysis to 6-formyltryptophan occurred slowly under neutral conditions with a first-order rate constant of 0.0039 min-1. This process, however, was accelerated by 10(4)-fold upon deprotonation of the indolyl nitrogen (N-1) at high pH. Tryptophanase did not detectably facilitate this hydrolysis reaction, since no protein-dependent conversion of the difluoromethyl group was detected. Instead, the enzyme accepted the fluorinated species as an analogue of tryptophan and catalyzed the corresponding formation of 6-(difluoromethyl)indole, pyruvate, and ammonium ion. Anionic intermediates are therefore not expected to form during the catalytic activation of the indolyl moiety. Instead, aromatic protonation likely promotes the release of indole during enzymatic degradation of tryptophan.

Site-specific and photo-induced alkylation of DNA by a dimethylanthraquinone-oligodeoxynucleotide conjugate.

A dialkyl-substituted anthraquinone derivative was synthesized and ligated to a sequence-directing oligodeoxynucleotide to examine its efficiency and specificity for cross-linking to complementary sequences of DNA. The anthraquinone appendage stabilized spontaneous hybridization of the target and probe sequences through non-covalent interactions, as indicated by thermal denaturation studies. Covalent modification of the target was induced by exposure to near UV light (lambda > 335 nm) to generate cross-linked duplexes in yields as great as 45%. Reaction was dependent on the first unpaired nucleotide extended beyond the duplex formed by association of the target and probe. A specificity of C > T > A = G was determined for modification at this position. The overall site and nucleotide selectivity seems to originate from the chemical requirements of cross-linking and does not likely reflect the dominant solution structure of the complex prior to irradiation.

Probing nucleic acid structure with nickel- and cobalt-based reagents.

The use of nickel and cobalt reagents is presented for characterizing the solvent exposure of guanine residues in DNA and RNA. These reagents promote guanine oxidation in the presence of a peracid such as monopersulfate, and the extent of reaction indicates the steric and electronic environment surrounding the N7 and aromatic face of this residue. Since oxidation does not itself perturb target structure or induce strand scission, it is coupled with fragmentation by treatment with piperidine (for smaller polynucleotides) or termination of primer extension (for larger polynucleotides).

Diamine preparation for synthesis of a water soluble Ni(II) salen complex.

A reliable and efficient synthesis of a Ni(II) salen complex useful in probing nucleic acid structure is described and illustrates a general approach for constructing cis diamines suitable for assembly into N2O2 Schiff base complexes. Two equivalents of an aryllithium reacted with 1,4-dimethylpiperazine-2,3-dione to form the symmetric alpha-dione. This material was then converted to its dioxime and reduced by TiCl4/NaBH4 to yield the meso-diamine. Condensation of the diamine and salicyladehyde, coordination of nickel and final methylation generated the desired water soluble and redox active complex.

The chemical modification of enzymatic specificity.

The chemical modification of enzymes has played and will continue to play an important role in probing the mechanism of enzyme activity. This technique can be utilized for identification of those individual amino acid residues responsible for the catalytic properties of the entire protein. In chemical modification experiments, changes in enzymatic specificity have been noted, but often not predicted. However, in recent years, rational approaches for the alteration of enzymatic properties have become feasible by means of site-specific mutagenesis and chemical methodology. In the first method, one amino acid can be replaced by a new one; in the second method, not only can new amino acid residues be introduced but also new catalytic entities (such as flavins) can be affixed to the protein molecule. Both methodologies are in their infancy, yet they represent a potentially powerful approach toward the design and synthesis of enzymes possessing new specificities.

Structural dependence of oligonucleotide photooxidation.

Oxidative photosensitization was used to characterize the conformational-dependent reactivity of various structures formed by oligonucleotides 14-15 nucleotides in length. The rate and product composition from a single hit process was analyzed using quantitative ion exchange chromatography under native and denaturing conditions. The primary damage incurred under aerobic acetone sensitization was base oxidation that, in turn, would induce strand scission upon a secondary treatment with piperidine. The reactive intermediates of this process were not consistent with diffusible radical species or singlet oxygen, as indicated by isotope and quenching studies. Derivatization was most likely initiated through a type I photoprocess with a direct interaction between DNA bases and excited state acetone preceding an irreversible oxidation step. This dominant reaction demonstrated no obvious sequence or site specificity for initial modification; the relative reactivity among the oligonucleotides did not correspond to any simple trend of base composition or near neighbor analysis. Likewise, the steric requirements of base modification allowed for similar rates of oxidation for single-strand, helical, and aberrant forms of DNA. Hybridization of the most reactive oligonucleotides, however, did suppress their relative single-strand vs double-strand reactivity by as much as fourfold.

Target-promoted alkylation of DNA.

Inducible and selective alkylation of DNA was accomplished under neutral conditions by use of a silyl-protected phenol that served as a precursor for a highly reactive quinone methide. As expected, addition of fluoride triggered reaction of a model compound, 3-(tert-butyldimethylsiloxy)-4-[(p-nitrophenoxy)methyl]benzamide, and its oligodeoxynucleotide conjugate. Surprisingly, the silyl phenol was also specifically yet more slowly activated by the environment of duplex DNA in the absence of fluoride. This alternative process was associated with the hybridization of probe and target strands, and single-stranded DNA was unable to induce a similar activation. Therefore, DNA appears to effect its own alkylation by promoting the formation of an electrophilic and nondiffusible intermediate.

2'-Deoxyguanosine reacts with a model quinone methide at multiple sites.

Quinone methides and related intermediates have been implicated in a range of beneficial and detrimental processes in biology and effectively alkylate a variety of cellular components despite the ubiquitous presence of water. As a prerequisite to understanding the origins of their specificity, the major products generated by DNA and its components with an unsubstituted ortho quinone methide under aqueous conditions were recently characterized [Pande, P., Shearer, J., Yang, J., Greenberg, W. A., and Rokita, S. E. (1999) J. Am. Chem. Soc. 121, 6773-6779]. Investigations currently focus on the complete range of derivatives formed by deoxyguanosine (dG) and guanine residues in duplex DNA through product isolation and structure determination using reversed-phase chromatography and a range of one and two-dimensional NMR techniques. Previous construction of a synthetic standard for dG alkylation is now shown to have yielded the N1-linked adduct rather than the N(2)-linked adduct. This later adduct has also now been characterized and confirmed to be the major product of reaction between the quinone methide and both duplex DNA and dG under neutral conditions. An N7 adduct of guanine has additionally been identified under these conditions and appears to result from spontaneous deglycosylation of the corresponding N7 adduct of dG. A combination of steric and electronic properties of duplex DNA likely contribute to the enhanced selectivity of the quinone methide for its guanine N(2) position (7.8:3.2:1.0 for adducts of N(2):N7:N1) relative to that of dG (4.7:3.5:1.0 for adducts of N(2):N7:N1).

3-fluoro-3-deoxycitrate: a probe for mechanistic study of citrate-utilizing enzymes.

The interaction of a novel fluorinated analogue of citrate, 3-fluoro-3-deoxycitrate (3-fluorocitrate), with the four known citrate-processing enzymes is described in this report. Three of the citrate-processing enzymes, citrate synthase, ATP citrate lyase, and citrate lyase, catalyze reversible aldol-type condensations. The fate of 3-fluorocitrate with each enzyme is uniquely related to their mechanisms of action. For citrate synthase, 3-fluorocitrate is a competitive inhibitor. 3-Fluorocitrate is a substrate for the carboxylate activation half-reaction catalyzed by ATP citrate lyase and induces a net ATPase action during conversion to 3-fluorocitryl-S-coenzyme A. Because of the unusual mechanism of citrate cleavage catalyzed by bacterial citrate lyase, 3-fluorocitrate is a mechanism-based inhibitor, acting at two points during turnover of the acetyl enzyme. The fourth citrate-processing enzyme, aconitase, does turn over 3-fluorocitrate catalytically. This enzyme, catalyzing a dehydration and rehydration of citrate, also catalyzes the elimination of HF from 3-fluorocitrate, yielding cis-aconitate and fluoride.

Nickel- and cobalt-dependent reagents identify structural features of RNA that are not detected by dimethyl sulfate or RNase T1.

Ribosomal 5S RNA presents a particular challenge to structural investigations since this polynucleotide is too large for complete NMR characterization but lacks significant tertiary structure to modulate, for example, diagnostic alkylation of guanine N7 by dimethyl sulfate. Nickel- and cobalt-dependent reagents that are sensitive to the N7 and aromatic face of guanine have now been applied to 5S rRNA (Xenopus lavis) and provide structural information that was not previously available from traditional chemical or enzymatic probes. Although G75 had repeatedly demonstrated an average reactivity with dimethyl sulfate and minimal reactivity with RNase T1, this residue was the major target of both metal-dependent reagents. Such reactivity provides crucial support for a structural model of loop E identified by prior physical, but not chemical, methods. Similarly, the tetraloop structure of loop D was more accurately reflected by the reactivity of G87 and G89 in the presence of the nickel reagent rather than in the presence of RNase T1. In addition, nickel-dependent modification of guanine residues surrounding the three-helix junction of loop A suggests an organization that is less compact than previously considered.