Studies of sulfate utilization by algae: 8. The ubiquity of sulfate reduction to thiosulfate

Cell-free extracts from several microorganisms, when prepared by methods originally devised for Chlorella pyrenoidosa (Emerson strain 3) and incubated anaerobically with ATP, Mg²⁺, and 2, 3-dimercaptopropan-1-ol, are capable of reducing sulfate-³⁵S to thiosulfate. These microorganisms include, in addition to C. pyrenoidosa (Emerson strain 3), several other strains of C. pyrenoidosa, Chlorella protothecoides, Chlorella vulgaris, Anacystis sp., Chlamydomonas reinhardi, Escherichia coli, Salmonella typhimurium, and baker's yeast. Three of these organisms, E. coli, S. typhimurium, and baker's yeast, were previously reported by others to reduce sulfate to sulfite. Moreover, three mutant strains of S. typhimurium (Ba-25, Ce-363, and Bc-482) previously reported by other workers to be unable to reduce sulfate to sulfite also cannot form thiosulfate, and one mutant strain (Cd-68) reportedly able to form sulfite can also form thiosulfate. Taken together, this suggests that thiosulfate-forming activity may be a common feature of sulfate-reducing systems, and it may be present in enzymatic systems previously thought to be forming sulfite. Reasonably conclusive identification of thiosulfate is provided by ion exchange chromatography and by paper electrophoresis; the ambiguities associated with other analytical methods are discussed.     

Studies of sulfate utilization by algae. 5. Identification of thiosulfate as a major Acid-volatile product formed by a cell-free sulfate-reducing system from chlorella

Separation of the products formed from sulfate-³⁵S by cell-free extracts of Chlorella pyrenoidosa (Emerson Strain 3) has permitted the identification of thiosulfate as a major product which yields acid-volatile radioactivity. The products formed, as separated by Dowex-1-nitrate chromatography, are qualitatively the same whether extracts at pH 7.0 (using TPNH as the reductant) or extracts at pH 9 [using 2,3-dimercaptopropan-1-ol, (BAL) as reductant] are employed. While thiosulfate can be separated without the addition of carrier, the inclusion of carrier improves the recovery. High concentrations of ATP which have been shown previously to inhibit the formation of acid-volatile radioactivity from radioactive sulfate, inhibit the formation of thiosulfate almost completely. Degradation of the thiosulfate formed at normal ATP concentrations reveals that most of the radioactivity is in the SO₃-sulfur of the molecule suggesting that the SH-sulfur is derived from the enzyme extracts. If carrier sulfite is present during thiosulfate formation from sulfate-³⁵S, radioactive sulfite is recovered at the expense of radioactive thiosulfate. Reconstruction experiments utilizing specifically-labeled thiosulfates indicate that radioactive sulfite formation is probably not the result of trapping a normal intermediate, but can be attributed to non-enzymatic exchange between labeled thiosulfate formed from sulfate and the non-radioactive sulfite added, suggesting that free sulfite is not an intermediate in thiosulfate formation from sulfate.     

Studies of Sulfate Utilization by Algae. 6. Adenosine-3'-Phosphate-5'-Phosphosulfate (PAPS) as an Intermediate in Thiosulfate Formation From Sulfate by Cell-Free Extracts of Chlorella

When cell-free preparations of Chlorella pyrenoidosa Chick (Emerson strain 3) form thiosulfate from labeled sulfate, another radioactive compound also appears. This compound has been isolated in quantity and is shown to be identical with adenosine-3′-phosphate-5′-phosphosulfate (PAPS) on the basis of its chromatographic and electrophoretic behavior, chemical composition, sensitivity to selective degradative enzymes, and its ability to serve as a substrate for rat liver aryl sulphotransferase. In addition, as expected for PAPS, the compound on mild acid treatment yields all of its radioactive sulfur as sulfate, and is converted to a compound identical with adenosine-3′,5′-diphosphate (PAP). Replacement of sulfate and ATP by this PAP³⁵S in the usual incubation mixture yields the same product, thiosulfate, which can be isolated as such or detected as acid-volatile radioactivity. This conversion of PAP³⁵S to thiosulfate still requires the addition of Mg²⁺ and a reductant such as 2,3-dimercaptopropan-1-ol (BAL). The cause of our previous result that high concentrations of ATP inhibit thiosulfate formation from sulfate can be ascribed to a small amount of PAP contaminating the ATP preparations, since PAP proves to be an exceedingly effective inhibitor of the conversion of PAP³⁵S to thiosulfate. Sulfate reduction to thiosulfate by Chlorella extracts is discussed and compared with similar systems from other organisms.     

Regulation of sulfate transport in filamentous fungi

Inorganic sulfate enters the mycelia of Aspergillus nidulans, Penicillium chrysogenum, and Penicillium notatum by a temperature-, energy-, pH-, ionic strength-, and concentration-dependent transport system (“permease”). Transport is unidirectional. In the presence of excess external sulfate, ATP sulfurylase-negative mutants will accumulate inorganic sulfate intracellularly to a level of about 0.04 m. The intracellular sulfate can be retained against a concentration gradient. Retention is not energy-dependent, nor is there any exchange between intracellular (accumulated) and extracellular sulfate. The sulfate permease is under metabolic control. Sulfur starvation of high methionine-grown mycelia results in about a 1000-fold increase in the specific sulfate transport activity at low external sulfate concentrations. l-Methionine is a metabolic repressor of the sulfate permease, while intracellular sulfate and possibly l-cysteine (or a derivative of l-cysteine) are feedback inhibitors. Sulfate transport follows hyperbolic saturation kinetics with a Michaelis constant (Km) value of 6 × 10⁻⁵ to 10⁻⁴m and a V_max (for maximally sulfurstarved mycelia) of about 5 micromoles per gram per minute. Refeeding sulfur-starved mycelia with sulfate or cysteine results in about a 10-fold decrease in the V_max value with no marked change in the Km. Azide and dinitrophenol also reduce the V_max.     

In vivo molybdate inhibition of sulfate transfer to porphyridium capsular polysaccharide

Active transport of exogenous sulfate into log phase cells of Porphyridium aerueineum followed Michaelis-Menten kinetics, and the apparent Km for sulfate transport is approximately 2.5 × 10⁻⁶m. Molybdate, also a group VI anion, is a competitive inhibitor of sulfate transport, and the inhibition is freely reversible. Once in the cell, molybdate depresses the rate of sulfate pool utilization by blocking sulfate transfer to polysaccharides destined for secretion to the cell surface. Specifically, molybdate inhibits the formation of adenosine 5′-phosphosulfate and in turn the formation of adenosine 3′-phosphate 5′-phosphosulfate, the activated donor for sulfate transfer reactions. Combined with the previous identification of adenosine 3′-phosphate 5′-phosphosulfate, this is taken as evidence that the adenosine 5′-phosphosulfate/adenosine 3′-phosphate 5′-phosphosulfate enzymatic sequence for sulfate activation and sulfate donor reactions is operating in Porphyridium. Thiosulfate is utilized as effectively as sulfate as both a sulfur source for growth and polysaccharide synthesis.     

Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.     

Some observations on the biosynthesis of the plant sulpholipid by Euglena gracilis

1. dl-Cysteine decreases the uptake of ³⁵SO₄²⁻ by Euglena gracilis but does not decrease the relative incorporation of the isotope into sulpholipid; cysteic acid, on the other hand, does not affect the uptake of ³⁵SO₄²⁻ but does dilute out its incorporation into the sulpholipid. 2. Both l-[³⁵S]cysteic acid and dl-+meso-[3-¹⁴C]cysteic acid appear almost exclusively in 6-sulphoquinovose. 3. Molybdate inhibits the incorporation of ³⁵SO₄²⁻ into sulpholipid but not its uptake into the cells; this suggests that adenosine 3′-phosphate 5′-sulphatophosphate may be concerned with the biosynthesis of sulpholipid, and it was shown to be formed by chloroplast fragments. 4. An outline scheme for sulpholipid biosynthesis based on these observations is discussed.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.     

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of ³⁵SO₄²⁻, and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using ³⁵SO₄²⁻, roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5′-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5′-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5′-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.     

Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H2

Twelve Tn5-induced mutants of Bradyrhizobium japonicum unable to grow chemoautotrophically with CO₂ and H₂ (Aut⁻) were isolated. Five Aut⁻ mutants lacked hydrogen uptake activity (Hup⁻). The other seven Aut⁻ mutants possessed wild-type levels of hydrogen uptake activity (Hup⁺), both in free-living culture and symbiotically. Three of the Hup⁻ mutants lacked hydrogenase activity both in free-living culture and as nodule bacteroids. The other two mutants were Hup⁻ only in free-living culture. The latter two mutants appeared to be hypersensitive to repression by oxygen, since Hup activity could be derepressed under 0.4% O₂. All five Hup⁻ mutants expressed both ex planta and symbiotic nitrogenase activities. Two of the seven Aut⁻ Hup⁺ mutants expressed no free-living nitrogenase activity, but they did express it symbiotically. These two strains, plus one other Aut⁻ Hup⁺ mutant, had CO₂ fixation activities 20 to 32% of the wild-type level. The cosmid pSH22, which was shown previously to contain hydrogenase-related genes of B. japonicum, was conjugated into each Aut⁻ mutant. The Aut⁻ Hup⁻ mutants that were Hup⁻ both in free-living culture and symbiotically were complemented by the cosmid. None of the other mutants was complemented by pSH22. Individual subcloned fragments of pSH22 were used to complement two of the Hup⁻ mutants.     

Achromobacter denitrificans Strain YD35 Pyruvate Dehydrogenase Controls NADH Production To Allow Tolerance to Extremely High Nitrite Levels

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO₂⁻) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO₂⁻-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO₂⁻ tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO₂⁻. Nitrite decreased the cellular NADH/NAD⁺ ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO₂⁻-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO₂⁻. These findings demonstrate a link between NO₂⁻ tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO₂⁻-tolerating mechanism in this strain.     

Regulation of Sulfate Assimilation in Plants : XIII. Assimilatory Sulfate Reduction during Ontogenesis of Primary Leaves of Phaseolus vulgaris L

The correlation between the extractable activities of three key enzymes of assimilatory sulfate reduction and the in vivo incorporation of ³⁵SO₄²⁻ into amino acids, proteins, and sulfolipids was investigated from greening to senescence in primary leaves of beans (Phaseolus vulgaris L.). The total extractable activity of ATP sulfurylase (EC 2.7.7.4) and of adenosine 5′-phosphosulfate sulfotransferase reached a maximum in the leaves of approximately 7- and 11-day-old seedlings, respectively. During senescence, there was a decrease in both enzyme activities. After approximately 17 days, no appreciable activities remained. In contrast, total O-acetyl-l-serine sulfhydrylase (EC 4.3.99.8) activity decreased to only approximately 50% of the maximal value during the same period. The in vivo incorporation of ³⁵SO₄²⁻ into amino acid and protein fractions showed a time-course similar to that of the total extractable adenosine 5′-phosphosulfate sulfotransferase activity. Both cysteine and sulfate markedly decreased during senescence. The total extractable activity of ribulosebisphosphate carboxylase (EC 4.1.1.39) was maximal in the primary leaves of 13-day-old seedlings, and approximately 40% of this value was still detectable after 17 days. Taken together with results from the literature, these results show that assimilatory sulfate reduction in primary leaves of P. vulgaris L. stops before CO₂ and nitrate assimilation.     

A Novel A3 Group Aconitase Tolerates Oxidation and Nitric Oxide

Achromobacter denitrificans YD35 is an NO₂⁻-tolerant bacterium that expresses the aconitase genes acnA3, acnA4, and acnB, of which acnA3 is essential for growth tolerance against 100 mm NO₂⁻. Atmospheric oxygen inactivated AcnA3 at a rate of 1.6 × 10⁻³ min⁻¹, which was 2.7- and 37-fold lower compared with AcnA4 and AcnB, respectively. Stoichiometric titration showed that the [4Fe-4S]²⁺ cluster of AcnA3 was more stable against oxidative inactivation by ferricyanide than that of AcnA4. Aconitase activity of AcnA3 persisted against high NO₂⁻ levels that generate reactive nitrogen species with an inactivation rate constant of k = 7.8 × 10⁻³ min⁻¹, which was 1.6- and 7.8-fold lower than those for AcnA4 and AcnB, respectively. When exposed to NO₂⁻, the acnA3 mutant (AcnA3Tn) accumulated higher levels of cellular citrate compared with the other aconitase mutants, indicating that AcnA3 is a major producer of cellular aconitase activity. The extreme resistance of AcnA3 against oxidation and reactive nitrogen species apparently contributes to bacterial NO₂⁻ tolerance. AcnA3Tn accumulated less cellular NADH and ATP compared with YD35 under our culture conditions. The accumulation of more NO by AcnA3Tn suggested that NADH-dependent enzymes detoxify NO for survival in a high NO₂⁻ milieu. This novel aconitase is distributed in Alcaligenaceae bacteria, including pathogens and denitrifiers, and it appears to contribute to a novel NO₂⁻ tolerance mechanism in this strain.     

A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase

Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3′-azido-2′,3′-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5′-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s⁻¹) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 µM) and chemistry forward and reverse reactions, 0.38 and 0.22 s⁻¹, respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing k_cat/K_m (7 × 10⁻⁶ μ M⁻¹ s⁻¹). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5′-triphosphate (8-oxo-dGTP) show this same phenomena.     

Energetics of Syntrophic Propionate Oxidation in Defined Batch and Chemostat Cocultures

Propionate consumption was studied in syntrophic batch and chemostat cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. The Gibbs free energy available for the H₂-consuming methanogens was <−20 kJ mol of CH₄⁻¹ and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >−10 kJ mol of propionate⁻¹. Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0.07 day⁻¹ and produced maximum biomass yields of 2.6 g mol of propionate⁻¹ and 7.6 g mol of CH₄⁻¹ for S. fumaroxidans and M. hungatei, respectively. The energy efficiency for syntrophic growth of S. fumaroxidans, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP⁻¹. However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown S. fumaroxidans was surprisingly low (0.14 kJ h⁻¹ mol of biomass C⁻¹) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.     

Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

The P_R promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that P_R has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of P_R showed that it uses a canonical −10 hexamer recognized by SigA, and mutants with mutations to the sequence 5′-TATAMT had the greatest activities. It does not contain a 5′-TGN-extended −10 sequence, although mutants with mutations creating an extended −10 sequence had substantially increased promoter activity. Mutations in the −35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the −35 hexamer differentially affected promoter activity, depending on the −10 and extended −10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout P_R generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter.     

Dynamics of Bacterial Sulfate Reduction in a Eutrophic Lake

Bacterial sulfate reduction in the surface sediment and the water column of Lake Mendota, Madison, Wis., was studied by using radioactive sulfate (³⁵SO₄²⁻). High rates of sulfate reduction were observed at the sediment surface, where the sulfate pool (0.2 mM SO₄²⁻) had a turnover time of 10 to 24 h. Daily sulfate reduction rates in Lake Mendota sediment varied from 50 to 600 nmol of SO₄²⁻ cm⁻³, depending on temperature and sampling date. Rates of sulfate reduction in the water column were 10³ times lower than that for the surface sediment and, on an areal basis, accounted for less than 18% of the total sulfate reduction in the hypolimnion during summer stratification. Rates of bacterial sulfate reduction in the sediment were not sulfate limited at sulfate concentrations greater than 0.1 mM in short-term experiments. Although sulfate reduction seemed to be sulfate limited below 0.1 mM, Michaelis-Menten kinetics were not observed. The optimum temperature (36 to 37°C) for sulfate reduction in the sediment was considerably higher than in situ temperatures (1 to 13°C). The response of sulfate reduction to the addition of various electron donors metabolized by sulfate-reducing bacteria in pure culture was investigated. The degree of stimulation was in this order: H₂ > n-butanol > n-propanol > ethanol > glucose. Acetate and lactate caused no stimulation.     

Reduction of Sulfur Compounds in the Sediments of a Eutrophic Lake Basin

Concentrations of various sulfur compounds (SO₄²⁻, H₂S, S⁰, acid-volatile sulfide, and total sulfur) were determined in the profundal sediments and overlying water column of a shallow eutrophic lake. Low concentrations of sulfate relative to those of acid-volatile sulfide and total sulfur and a decrease in total sulfur with sediment depth implied that the contribution of dissimilatory sulfur reduction to H₂S production was relatively minor. Addition of 1.0 mM Na₂³⁵SO₄ to upper sediments in laboratory experiments resulted in the production of H₂³⁵S with no apparent lag. Kinetic experiments with ³⁵S demonstrated an apparent K_m of 0.068 mmol of SO₄²⁻ reduced per liter of sediment per day, whereas tracer experiments with ³⁵S indicated an average turnover time of the sediment sulfate pool of 1.5 h. Total sulfate reduction in a sediment depth profile to 15 cm was 15.3 mmol of sulfate reduced per m² per day, which corresponds to a mineralization of 30% of the particulate organic matter entering the sediment. Reduction of ³⁵S⁰ occurred at a slower rate. These results demonstrated that high rates of sulfate reduction occur in these sediments despite low concentrations of oxidized inorganic compounds and that this reduction can be important in the anaerobic mineralization of organic carbon.     

Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation

This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9₂₅-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9₂₅-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5′-product terminating in a 2′,3′-phosphodiester and a 3′-product terminating in a 5′-OH. Under single turnover conditions at 24°C, 9₂₅-11t displays a maximum first-order rate constant, k_cat, of 0.037 min⁻¹ and a catalytic efficiency, k_cat/K_m, of 5.3 × 10⁵ M⁻¹ min⁻¹. The measured value of k_cat under catalyst excess conditions agrees with the value of k_cat observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9₂₅-11t was gauged in terms of k_cat values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k_cat values by a factor of 4 to 250, indicating that 9₂₅-11t displays significant sequence specificity despite the lack of an apparent Watson–Crick base-pairing scheme for recognition.