Is beta-poly(L-malate) synthesis catalysed by a combination of beta-L-malyl-AMP-ligase and beta-poly(L-malate) polymerase?

beta-Poly(L-malate) is supposed to function in the storage and transport of histones, DNA polymerases and other nuclear proteins in the giant syncytical cells (plasmodia) of myxomycetes. Here we report on the biosynthesis of [14C]beta-poly(L-malate) from injected L-[14C]malate in the plasmodium of Physarum polycephalum. The effects of KCN, arsenate, adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, guanosine 5'-(beta, gamma-methylene)triphosphate, desulfo coenzyme A and phenylarsinoxid on beta-poly(L-malate) synthesis were studied after their coinjection with L-[14C]malate. The synthesis was not affected by KCN or desulfo coenzyme A, but was blocked by arsenate and adenosine 5'-(alpha,beta-methylene)triphosphate. The plasmodium lysate catalysed an L-malate-dependent ATP-[32P]pyrophosphate exchange, but was devoid of beta-poly(L-malate) synthetic activity under all experimental conditions tested. The results suggested an extramitochondrial synthesis of beta-poly(L-malate), involving the polymerization of beta-L-malyl-AMP. It is assumed that the lack of synthesis in the lysate is caused by the inactivation of beta-poly(L-malate) polymerase involving a cell injury kinase pathway. Because injected guanosine 5'-(beta, gamma-methylene)triphosphate blocks the synthesis, the injury signal is likely to be GTP dependent.     

Probing DNA single strands for single-base bulges with neocarzinostatin chromophore.

Neocarzinostatin chromophore (NCS-Chrom) induces strong cleavage at a single site (C3) in the single-stranded and 5' (32)P-end-labeled 13-mer GCCAGATTTGAGC in a reaction dependent on a thiol. By contrast, in the duplex form of the same 13-mer, strand cleavage occurs only at the T and A residues, and C3 is not cleaved. To determine the minimal structural requirement(s) for C3 cleavage in the single-stranded oligomer, several deletions and mutations were made in the 13-mer. A 10-mer (GCCAGAGAGC) derived from the 13-mer by deletion of the three T residues was also cleaved exclusively at C3 by NCS-Chrom, generating fragments having 5' phosphate ends. That the cleavage at C3 is initiated by abstraction of its 5' hydrogen is confirmed in experiments using 3' (32)P-end-labeled 10-mer. The competent 13-mer and 10-mer were assigned hairpin structures with a stem loop and a single bulged out A base, placing C3 across from and 3' to the bulge. Removal of the bulged A base from the 13-mer and the 10-mer resulted in complete loss of cutting activity, proving that it is the essential determinant in competent substrates. Studies of thiol post-activated NCS-Chrom binding to the DNA oligomers show that the drug binds to the bulge-containing 13-mer (K(d) = 0.78 microM) and the 10-mer (K(d) = 1.11 microM), much more strongly than to the 12-mer (K(d) = 20 microM) and the 9-mer (K(d) = 41 microM), lacking the single-base bulge. A mutually induced-fit between NCS-Chrom and the oligomer resulting in optimal stabilization of the drug-DNA complex is proposed to account for the site-specific cleavage at C3. These studies establish the usefulness of NCS-Chrom as a probe for single-base bulges in DNA.     

A role for the passage helix in the DNA cleavage reaction of eukaryotic topoisomerase II. A two-site model for enzyme-mediated DNA cleavage.

Eukaryotic topoisomerase II is capable of binding two separate nucleic acid helices prior to its DNA cleavage and strand passage events (Zechiedrich, E. L., and Osheroff, N (1990) EMBO J. 9, 4555-4562). Presumably, one of these helices represents the helix that the enzyme cleaves (i.e. cleavage helix), and the other represents the helix that it passes (i.e. passage helix) through the break in the nucleic acid backbone. To determine whether the passage helix is required for reaction steps that precede the enzyme's DNA strand passage event, interactions between Drosophila melanogaster topoisomerase II and a short double-stranded oligonucleotide were assessed. These studies employed a 40-mer that contained a specific recognition/cleavage site for the enzyme. The sigmoidal DNA concentration dependence that was observed for cleavage of the 40-mer indicated that topoisomerase II had to interact with more than a single oligonucleotide in order for cleavage to take place. Despite this requirement, results of enzyme DNA binding experiments indicated no binding cooperativity for the 40-mer. These findings strongly suggest a two-site model for topoisomerase II action in which the passage and the cleavage helices bind to the enzyme independently, but the passage helix must be present for efficient topoisomerase II-mediated DNA cleavage to occur.     

Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.

The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically.     

Enhancing sequence-specific cleavage of RNA within a duplex region: incorporation of 1,3-propanediol linkers into oligonucleotide conjugates of serinol-terpyridine.

The syntheses and RNA cleavage efficiencies of a new series of oligonucleotide conjugates of Cu(II)-serinol-terpyridine and 1,3-propanediol are reported. These reagents, termed ribozyme mimics, were designed such that they would yield multiple unpaired RNA residues directly opposite the site of the RNA cleavage catalyst upon ribozyme mimic-RNA duplex formation. This design effect was implemented using the 1,3-propanediol linker 3, which mimics the three-carbon spacing between the 5'- and 3'-hydroxyls of a natural nucleotide. Incorporation of one or more of these 1,3-propanediol linkers at positions directly adjacent to the serinol-terpyridine modification in the ribozyme mimic DNA strand resulted in cleavage at multiple phosphates in a complementary 31-mer RNA target sequence. The linkers effectively created artificial mismatches in the RNA-DNA duplexes, rendering the opposing RNA residues much more susceptible to cleavage via the transesterification/hydrolysis pathway. The RNA cleavage products produced by the various mimics correlated directly with the number and locations of the linkers in their DNA strands, and the most active ribozyme mimic in the series exhibited multiple turnover in the presence of excess 31-mer RNA target.     

Effect of DNA on poly (ADP-ribose) glycohydrolase and the degradation of histone H1-poly (ADP-ribose) complex from HeLa cell nuclei.

A poly(ADP-ribose)-H1 histone complex has been isolated from HeLa cell nuclei incubated with NAD. The rate of poly(ADP-ribose) glycohydrolase catalyzed hydrolysis of the polymer in the complex is only 1/9 that of free poly(ADP-ribose), indicating that the polymer is in a protected environment within the complex. Comparison of the rate of hydrolysis of free poly(ADP-ribose) in the presence or absence of H1 to that in the complex synthesized de novo indicates a specific mode of packaging of the complex. This is further indicated by the fact that alkaline dissociation of the complex followed by neutralization markedly exposes the associated poly(ADP-ribose) to the glycohydrolase. The complex also partially unfolds when it binds to DNA as evidenced by a 2-fold increase in the rate of glycolytic cleavage of poly(ADP-ribose). This effect of DNA is not due to a stimulation of the glycohydrolase per se since hydrolysis of free polymer by the enzyme is strongly inhibited by DNA, especially single-stranded DNA. Inhibition of glycohydrolase by DNA results from the binding of the enzyme to DNA and conditions which decrease this binding (increased ionic strength or addition of histone H1 which competes for DNA binding) relieve the DNA inhibition.     

Development of peptide substrates for trypsin based on monomer/excimer fluorescence of pyrene

An assay using fluorogenic peptides based on the monomer/excimer fluorescence features of pyrene was developed to measure the proteolytic activity of trypsin, a serine protease. Two pyrene moieties were incorporated into the respective N- and C-terminus of the peptides as (pyrene)-C-Xaa-C-(pyrene), where Xaa represents amino acid residues of 5-, 6-, 7-, or 8-mer containing the cleavage site of trypsin. The proteolytic cleavage of the substrates led to an increase in monomer fluorescence and a decrease in excimer fluorescence of pyrene. Kinetic parameters (k(cat) and K(m)) for the enzymatic hydrolysis of the substrates were successfully determined. The parameters are dependent on the chain length of the substrate and optimal catalytic activity was obtained with substrates that consisted of 9 or 10 amino acid residues. The present assay system is sensitive and the preparation of the substrate is very simple. We suggest that this method may be suitable for high-throughput screening and also applicable to the characterization of other proteases.     

NMR studies of A.C mismatches in DNA dodecanucleotides at acidic pH. Wobble A(anti).C(anti) pair formation

Two-dimensional proton NMR studies were undertaken on the d(C-G-A-G-A-A-T-T-C-C-C-G) duplex (designated A.C 12-mer) where the A at the mismatch site is flanked by G residues and the d(C-G-C-G-A-A-T-T-C-A-C-G) duplex (designated C.A 12-mer) where the A at the mismatch site is flanked by C residues in an attempt to elucidate the role of flanking base pairs on the structure of the A.C mismatch. The exchangeable and nonexchangeable proton spectra of these two dodecanucleotides have been completely characterized by two-dimensional nuclear Overhauser enhancement (NOE) experiments in H2O and D2O solution at acidic pH. The NOE distance connectivities demonstrate that both A and C at the mismatch site are stacked into a right-handed helix between flanking G.C base pairs and exhibit anti-glycosidic torsion angles. The proton chemical shifts and NOE patterns are consistent with Wobble A.C pairing for the A.C 12-mer and C.A 12-mer duplexes in solution and demonstrate that the A.C mismatches introduce local conformational perturbations that do not extend to the central AATT segment. We detect that amino protons of adenosine (approximately 9.2 ppm) but not of cytidine at the A.C mismatch site in both duplexes on lowering the pH below 6.     

Plasticity in protein-peptide recognition: crystal structures of two different peptides bound to concanavalin A

The structures of concanavalin A (ConA) in complex with two carbohydrate-mimicking peptides, 10-mer (MYWYPYASGS) and 15-mer (RVWYPYGSYLTASGS) have been determined at 2.75 A resolution. In both crystal structures four independent peptide molecules bind to each of the crystallographically independent subunits of ConA tetramer. The peptides exhibit small but significant variability in conformations and interactions while binding to ConA. The crystal structure of another similar peptide, 12-mer (DVFYPYPYASGS), in complex with ConA has been determined (Jain, D., K. J. Kaur, B. Sundaravadivel, and D. M. Salunke. 2000. Structural and functional consequences of peptide-carbohydrate mimicry. J. Biol. Chem. 275:16098-16102). Comparison of the three complexes shows that the peptides bind to ConA at a common binding site, using different contacting residues and interactions depending on their sequence and the local environment at the binding site. The binding is also optimized by corresponding plasticity of the peptide binding site on ConA. The diversity in conformation and interactions observed here are in agreement with the structural leeway concerning plasticity of specific molecular recognition in biological processes. The adaptability of peptide-ConA interactions may also be correlated with the carbohydrate-mimicking property of these peptides.     

Activity of purified hepatitis C virus protease NS3 on peptide substrates.

The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation.     

Topography of the interaction of recA protein with single-stranded deoxyoligonucleotides

recA protein, in the presence of adenosine 5'-(gamma-thio)triphosphate, formed stable complexes with single-stranded deoxyoligonucleotides between 9 and 20 residues in length but not with those 8-residues long. The binding of recA protein to a 15-mer and 20-mer completely protected the sugar-phosphate backbone of the nucleic acid from digestion by pancreatic deoxyribonuclease I and protected the 5'-terminal phosphate from cleavage by calf intestinal alkaline phosphatase. Ethylation of the phosphate backbone at any position by ethylnitrosourea blocked the binding of recA protein to the 15-mer but not to the 20-mer. Ethylation of phosphates near the ends of the 15-mer interfered less, suggesting a minimum binding site requirement. In contrast to the protection of the nucleic acid backbone, recA protein did not protect the N-7 position of guanine or the N-3 position of adenine from methylation by dimethyl sulfate, but rather enhanced the methylation of guanine. These results indicate that recA protein binds primarily to the phosphate backbone of single-stranded DNA, leaving the bases free for homologous pairing. We present a model for the organization of the presynaptic filament.     

Binding of nucleic acids to E. coli RNase HI observed by NMR and CD spectroscopy.

To clarify the mechanism by which the RNA portion of a DNA/RNA hybrid is specifically hydrolyzed by ribonuclease H (RNase H), the binding of a DNA/RNA hybrid, a DNA/DNA duplex, or an RNA/RNA duplex to RNase HI from Escherichia coli was investigated by 1H-15N heteronuclear NMR. Chemical shift changes of backbone amide resonances were monitored while the substrate, a hybrid 9-mer duplex, a DNA/DNA 12-mer duplex, or an RNA/RNA 12-mer duplex was titrated. The amino acid residues affected by the addition of each 12-mer duplex were almost identical to those affected by the substrate hybrid binding, and resided close to the active site of the enzyme. The results reveal that all the duplexes, hybrid-, DNA-, and RNA-duplex, bind to the enzyme. From the linewidth analysis of the resonance peaks, it was found that the exchange rates for the binding were different between the hybrid and the other duplexes. The NMR and CD data suggest that conformational changes occur in the enzyme and the hybrid duplex upon binding.     

Interaction of the adenovirus proteinase with protein cofactors with high negative charge densities

The interactions of the human adenovirus proteinase (AVP) with polymers with high negative charge densities were characterized. AVP utilizes two viral cofactors for maximal enzyme activity (k(cat)/K(m)), the 11-amino acid peptide from the C-terminus of virion precursor protein pVI (pVIc) and the viral DNA. The viral DNA stimulates covalent AVP-pVIc complexes (AVP-pVIc) as a polyanion with a high negative charge density. Here, the interactions of AVP-pVIc with different polymers with high negative charge densities, polymers of glutamic acid (polyE), were characterized. The rate of substrate hydrolysis by AVP-pVIc increased with increasing concentrations of polyE. At higher concentrations of polyE, the increase in the rate of substrate hydrolysis approached saturation. Although glutamic acid did not stimulate enzyme activity, glutamic acid and NaCl could displace DNA from AVP-pVIc.(DNA) complexes; the K(i) values were 230 and 329 nM, respectively. PolyE binds to the DNA binding site on AVP-pVIc as polyE and DNA compete for binding to AVP-pVIc. The equilibrium dissociation constant for 1.3 kDa polyE binding to AVP-pVIc was 56 nM. On average, one molecule of AVP-pVIc binds to 12 residues in polyE. Comparison of polyE and 12-mer single-stranded DNA interacting with AVP-pVIc revealed the binding constants are similar, as are the Michaelis-Menten constants for substrate hydrolysis. The number of ion pairs formed upon the binding of 1.3 kDa polyE to AVP-pVIc was 2, and the nonelectrostatic change in free energy upon binding was -6.5 kcal. These observations may be physiologically relevant as they infer that AVP may bind to proteins that have regions of negative charge density. This would restrict activation of the enzyme to the locus of the cofactor within the cell.     

Epithelial sodium channel (ENaC) is multi-ubiquitinated at the cell surface

MMP-2 (matrix metalloproteinase 2) contains a CBD (collagen-binding domain), which is essential for positioning gelatin substrate molecules relative to the catalytic site for cleavage. Deletion of the CBD or disruption of CBD-mediated gelatin binding inhibits gelatinolysis by MMP-2. To identify CBD-binding sites on type I collagen and collagen peptides with the capacity to compete CBD binding of gelatin and thereby inhibit gelatinolysis by MMP-2, we screened a one-bead one-peptide combinatorial peptide library with recombinant CBD as bait. Analyses of sequences from the CBD-binding peptides pointed to residues 715-721 in human alpha1(I) collagen chain as a binding site for CBD. A peptide (P713) including this collagen segment was synthesized for analyses. In SPR (surface plasmon resonance) assays, the CBD and MMP-2(E404A), a catalytically inactive MMP-2 mutant, both bound immobilized P713 in a concentration-dependent manner, but not a scrambled control peptide. Furthermore, P713 competed gelatin binding by the CBD and MMP-2(E404A). In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2DeltaCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited approximately 90% of the MMP-2 gelatin cleavage, but less than 20% of the MMP-2 activity on a peptide substrate (NFF-1) which does not require the CBD for cleavage. Confirming the specificity of the inhibition, P713 did not alter MMP-2DeltaCBD or MMP-8 activities. These experiments identified a CBD-binding site on type I collagen and demonstrated that a corresponding synthetic peptide can inhibit hydrolysis of type I and IV collagens by competing CBD-mediated gelatin binding to MMP-2.     

Plasmid DNA cleavage by MunI restriction enzyme: single-turnover and steady-state kinetic analysis

Mutational analysis has previously indicated that D83 and E98 residues are essential for DNA cleavage activity and presumably chelate a Mg2+ ion at the active site of MunI restriction enzyme. In the absence of metal ions, protonation of an ionizable residue with a pKa > 7.0, most likely one of the active site carboxylates, controls the DNA binding specificity of MunI [Lagunavicius, A., Grazulis, S., Balciunaite, E., Vainius, D., and Siksnys, V. (1997) Biochemistry 36, 11093-11099.]. Thus, competition between H+ and Mg2+ binding at the active site of MunI presumably plays an important role in catalysis/binding. In the present study we have identified elementary steps and intermediates in the reaction pathway of plasmid DNA cleavage by MunI and elucidated the effect of pH and Mg2+ ions on the individual steps of the DNA cleavage reaction. The kinetic analysis indicated that the multiple-turnover rate of plasmid cleavage by MunI is limited by product release throughout the pH range 6.0-9.3. Quenched-flow experiments revealed that open circle DNA is an obligatory intermediate in the reaction pathway. Under optimal reaction conditions, open circle DNA remains bound to the MunI; however it is released into the solution at low [MgCl2]. Rate constants for the phoshodiester bond hydrolysis of the first (k1) and second (k2) strand of plasmid DNA at pH 7.0 and 10 mM MgCl2 more than 100-fold exceed the kcat value which is limited by product dissociation. The analysis of the pH and [Mg2+] dependences of k1 and k2 revealed that both H+ and Mg2+ ions compete for the binding to the same residue at the active site of MunI. Thus, the decreased rate of phosphodiester hydrolysis by MunI at pH < 7.0 may be due to the reduction of affinity for the Mg2+ binding at the active site. Kinetic analysis of DNA cleavage by MunI yielded estimates for the association-dissociation rate constants of enzyme-substrate complex and demonstrated the decreased stability of the MunI-DNA complex at pH values above 8.0.     

Preferential binding to DNA sequences of peptides related to a novel XPRK motif

Two dodecapeptide amines: (WPRK)(3)NH(2)[WR-12] and (YPRK)(3)NH(2)[YR-12], and a 30-mer polypeptide amide (SP-30) were synthesized by solid-phase peptide methodology. DNase I footprinting studies on a 117-mer DNA showed that WR-12 and YR-12 bind selectively to DNA sequences in a manner similar to SP-30 which has a repeating SPK(R)K sequence. The most distinctive blockages seen with all three peptides occur at positions 26-30, 21-24 and 38-45 around sequences 5'-GAATT-3', 5'-TAAT-3' and 5'-AAAACGAC-3', respectively. However, it appears that YR-12 is better able to extend its recognition site to include CG pairs than is SP-30. At low concentrations YR-12 was able to induce enhanced rates of DNase I cleavage at regions surrounding some of its binding sites. To obtain further quantitative data supplementary to the footprinting work, equilibrium binding experiments were performed in which the binding of the two peptides to six decanucleotide duplexes was compared. Scatchard analyses indicated that WR-12 may be more selective for oligomers containing runs of consecutive purines or pyrimidines. On the other hand, YR-12 binds better to d(CTTAGACGTC)- d(GACGTCTAAG) than to the other oligomer duplexes, denoting selectivity for evenly distributed C/G and A/T sequences.     

Conformational selection and collagenolysis in Type III collagen

Matrix metalloproteases (MMPs) cleave native collagen at a single site despite the fact that collagen contains more than one scissile bond that can, in principle, be cleaved. For peptide bond hydrolysis to occur at one specific site, MMPs must (1) localize to a region near the unique scissile bond, (2) bind residues at the catalytic site that form the scissile bond, and (3) hydrolyze the corresponding peptide bond. Prior studies suggest that for some types of collagen, binding of noncatalytic MMP domains to amino acid sequences in the vicinity of the true cleavage site facilitates the localization of collagenases. In the present study, our goal was to determine whether binding to the catalytic site also plays a role in determining MMP specificity. To investigate this, we computed the conformational free energy landscape of Type III collagen at each potential cleavage site. The free energy profiles suggest that although all potential cleavage sites sample unfolded states at relatively low temperatures, the true cleavage site samples structures that are complementary to the catalytic site. By contrast, potential cleavage sites that are not cleaved sample states that are relatively incompatible with the MMP active site. Furthermore, our findings point to a specific role for arginine residues in modulating the structural stability of collagen near the collagenase cleavage site. These data imply that locally unfolded potential cleavage sites in Type III collagen sample distinct unfolded ensembles, and that the region about the true collagenase cleavage site samples states that are most complementary to the MMP active site. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Detection and characterization of two ATP-dependent conformational changes in proteolytically inactive Escherichia coli Lon mutants by stopped flow kinetic techniques

Lon is an ATP dependent serine protease responsible for degrading denatured, oxidatively damaged and certain regulatory proteins in the cell. In this study we exploited the fluorescence properties of a dansylated peptide substrate (S4) and the intrinsic Trp residues in Lon to monitor peptide interacting with the enzyme. We generated two proteolytically inactive Lon mutants, S679A and S679W, where the active site serine is mutated to an Ala and Trp residue, respectively. Stopped-flow fluorescence spectroscopy was used to identify key enzyme intermediates generated along the reaction pathway prior to peptide hydrolysis. A two-step peptide binding event is detected in both mutants, where a conformational change occurs after a rapid equilibrium peptide binding step. The Kd for the initial peptide binding step determined by kinetic and equilibrium binding techniques is approximately 164 micromolar and 38 micromolar, respectively. The rate constants for the conformational change detected in the S679A and S679W Lon mutants are 0.74 +/- 0.10 s(-1) and 0.57 +/- 0.10 s(-1), respectively. These values are comparable to the lag rate constant determined for peptide hydrolysis (klag approximately 1 s(-1)) [Vineyard, D., et al. (2005) Biochemistry 45, 4602-4610]. Replacement of the active site Ser with Trp (S679W) allows for the detection of an ATP-dependent conformational change within the proteolytic site. The rate constant for this conformational change is 7.6 +/- 1.0 s(-1), and is essentially identical to the burst rate constant determined for ATP hydrolysis under comparable reaction conditions. Collectively, these kinetic data support a mechanism by which the binding of ATP to an allosteric site on Lon activates the proteolytic site. In this model, the energy derived from the binding of ATP minimally supports peptide cleavage by allowing peptide substrate access to the proteolytic site. However, the kinetics of peptide cleavage are enhanced by the hydrolysis of ATP.     

DNA hydrolysis and oxidative cleavage by metal-binding peptides tethered to rhodium intercalators.

With the goal of developing artificial nucleases for DNA hydrolysis, metal-coordinating peptides have been tethered to a DNA-intercalating rhodium complex to deliver metal ions to the sugar-phosphate backbone. The intercalator, [Rh(phi)(2)bpy']Cl(3) [phi = 9,10-phenanthrenequinone diimine; bpy' = 4-(butyric acid)-4'-methyl-2,2'-bipyridine], provides DNA binding affinity, and a metal-binding peptide contributes reactivity. This strategy for DNA hydrolysis is a general one, and zinc(II)-promoted cleavage has been demonstrated for two widely different tethered metallopeptides. An intercalator coupled with a de novo-designed alpha helix containing two histidine residues has been demonstrated to cleave both supercoiled plasmid and linear DNA substrates. Mutation of this peptide confirms that the two histidine residues are essential for Zn(2+) binding and cleavage. Zinc(II)-promoted cleavage of supercoiled plasmid has also been demonstrated with an intercalator-peptide conjugate containing acidic residues and modeled after the active site of the BamHI endonuclease. Other redox-active metals, such as copper, have been delivered to DNA with our intercalator-peptide conjugates to effect oxidative chemistry. Copper cleavage experiments and photocleavage experiments with [Rh(phi)(2)bpy'](3+) complement the hydrolysis studies and provide structural information about the interactions between the tethered metallopeptides and DNA. Variation of the rhodium intercalator was also explored, but with a mismatch-specific intercalator, no site-specific hydrolysis was found. These experiments, in which the peptide, the metal cation, and the intercalator components of the conjugate are each varied, illustrate some of the issues involved in creating an artificial nuclease with DNA intercalators and metallopeptides.     

Simple and Rapid Enzymatic Method for the Synthesis of Single-Strand Oligonucleotides Containing Trifluorothymidine

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5′-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5′-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.