Nitrogen 15 tracer studies on the pathway of denitrification in Pseudomonas aeruginosa.

The pathway of anaerobic reduction of nitrite to nitrogen gas (N2) by cell suspensions of the denitrifier, Pseudomonas aeruginosa, was studied using the techniques of gas chromatography and mass spectrometry. While release of nitrous oxide (N2O) is not normally detected during the reduction of nitrite to N2 by this organism, 15N from [15N]nitrite nevertheless can be trapped quantitatively as 15N2O in a pool of added N2O. In such experiments the abundance of 15N in N2O always exceeds that in product N2, consistent with the absence of a major reductive route from nitrite to N2 which by-passes N2O. During the reduction of a mixture of [15N]nitrite and nitric oxide (NO), 15NO produced at most only in trace amounts. The final products are chiefly 15N2 and 14N2 with only a small fraction of the scrambled product, 14N15N. Much of the 14N15N can be accounted for as an artifact caused by traces of molecular oxygen, which promote the conversion of NO to nitrite by autooxidation and thereby degrade slightly the isotopic purity of [15N]nitrite. Nitrous oxide shows all the properties of a free obligatory intermediate during the denitrification of nitrite to N2 by P. aeruginosa, whereas NO does not. The inability to trap 15NO in a pool of NO indicates that NO is not a free obligatory intermediate in the reduction of nitrite. The small mole fractions of 14N15N produced from a mixture of [15N]nitrite and NO require that the main reductive pathways for these nitrogen oxides cannot share any freely diffusible mono-nitrogen intermediate in common. The simplest interpretation is that nitrite and NO are denitrified by separate pathways, at least prior to the formation of the first bi-nitrogen compound.     

Evidence of an associative intermediate on the myoglobin refolding pathway.

Time-resolved small-angle x-ray scattering using the stopped-flow method has been applied successfully to investigate the refolding of myoglobin. This is the only method to date that yields direct information on protein physical dimensions during the folding process. It has the potential to detect and probe important processes, such as protein compaction and association, on a millisecond time scale. Initial experiments were performed with horse myoglobin denatured in high concentrations of urea. The denatured protein was diluted rapidly into a buffer containing no urea or low concentrations of urea. The time-course of the forward-scattered intensity shows a decrease in amplitude which is clearly not engendered by the compaction of the protein, but does correspond well to a dimer dissociation process. Initial and final radii of gyration correspond well to a dimer and a monomer, respectively. Kratky plots of the initial and final states also support the transient dimerization model. The apparent dissociation rate constant was obtainable directly from the data. An association rate constant and an equilibrium constant could be estimated. The dimerizing intermediate is speculated to be a globular non-native state with an exposed hydrophobic surface.     

Stability and folding studies of the N-domain of troponin C. Evidence for the formation of an intermediate

We report here on the stability and folding of the 91 residue alpha-helical F29W N-terminal domain of chicken skeletal muscle troponin C (TnC(1-91)F29W), the thin filament calcium-binding component. Unfolding was monitored by differential scanning calorimetry, circular dichroism, and intrinsic fluorescence spectroscopy using urea, pH, and temperature as denaturants, in the absence and in the presence of calcium. The unfolding of TnC(1-91)F29W was reversible and did not follow a two-state transition, suggesting that an intermediate may be present during this reaction. Our results support the hypothesis that intermediates are likely to occur during the folding of small proteins and domains. The physiological significance of the presence of an intermediate in the folding pathway of troponin C is discussed.     

Real-time NMR studies on a transient folding intermediate of barstar.

The refolding of barstar, the intracellular inhibitor of barnase, is dominated by the slow formation of a cis peptidyl prolyl bond in the native protein. The triple mutant C40/82A P27A in which two cysteine residues and one trans proline were replaced by alanine was used as model system to investigate the kinetics and structural consequences of the trans/cis interconversion of Pro48. One- and two-dimensional real-time NMR spectroscopy was used to follow the trans/cis interconversion after folding was initiated by rapid dilution of the urea denatured protein. Series of 1H, 15N HSQC spectra acquired with and without the addition of peptidyl prolyl isomerase unambiguously revealed the accumulation of a transient trans-Pro48 intermediate within the dead time of the experiment. Subtle chemical shift differences between the native state and the intermediate spectra indicate that the intermediate is predominantly native-like with a local rearrangement in the Pro48 loop and in the beta-sheet region including residues Tyr47, Ala82, Thr85, and Val50.     

The kinetic and isotopic competence of nitric oxide as an intermediate in denitrification

Rates of NO uptake by five denitrifying bacteria were estimated by NO-electrode and gas chromatography methods under conditions of rather low cell densities and [NOaq]. The rates so measured, VmaxNO, represent lower limits for the true value of that parameter, but nevertheless exceed Vmax for nitrite uptake, VmaxNi, by a factor of two typically. Previous estimates under suboptimal conditions had placed VmaxNO at 0.3-0.5 of VmaxNi (St. John, R. T., and Hollocher, T. C. (1977) J. Biol. Chem. 252, 212-218; Garber, E. A. E., and Hollocher, T.C. (1981) J. Biol. Chem. 256, 5459-5465). The steady-state [NOaq] during denitrification of nitrite by nitrate-grown cells was less than or equal to 1 microM. The above observations, taken with a recent direct estimate for the KmNO for NO uptake of 0.4 microM (Zafiriou, O. C., Hanley, Q. S., and Snyder, G. (1989) J. Biol. Chem. 264, 5694-5699), would allow NO to be a free intermediate between nitrite and N2O with steady-state concentrations of less than or equal to 0.4 microM. As the result of special conditions during cell growth or differential inhibition by azide, it was possible to establish systems that accumulated NO during denitrification of nitrite. In all such cases, VmaxNO less than VmaxNi, and the time required to reach the maximum [NOaq] corresponded closely to the time needed to exhaust the nitrite. A semiquantitative isotope experiment with Paracoccus denitrificans demonstrated the trapping of 15NO from 15NO2- in a pool of NOaq. A quantitative isotope method using low densities of the same bacterium showed that 15N from 15NO2- and 14N from NOg combine randomly to form N2O during the simultaneous denitrification of 15NO2- and NO. The result requires that the pathways from nitrite and NO share a common mononitrogen intermediate. Results to the contrary obtained at high cell densities (first two references cited above) are now believed to have been due to technical artifacts. The present results are consistent with the view that NO is under kinetic control as a free intermediate in denitrification and serve to remove previously imagined constraints on this view.     

15N tracer studies on the reduction of nitrite by the purified dissimilatory nitrite reductase of Pseudomonas aeruginosa. Evidence for direct production of N2O without free NO as an intermediate

Nitrite reductase (cytochrome c,d1) was purified from Pseudomonas aeruginosa. In the presence of the reducing system, ascorbate-N,N,N',N'-tetramethylphenyl-enediamine, which alone had no ability to reduce nitrite or NO at pH 7.5, the enzyme catalyzed the reduction of nitrite to NO and N2O as major and minor products, respectively, as determined by gas chromatography-mass spectrometry. The rate of reduction of NO to N2O was considerably lower than the rate of reduction of nitrite to N2O and might be zero. The N2O produced in a system containing [15N]nitrite and natural NO was more highly enriched in 15N than was the NO pool and, in this regard, closely resembled the enrichment of the nitrite pool. The amount of 14N in the NO pool changed little, if any, as the result of enzymatic processes. For the enzyme, free NO seems not to be an intermediate between nitrite and N2O, just as was found by this laboratory for certain intact denitrifying bacteria. The results are consistent with reduction of nitrite to enzyme-bound NO, which can partition between release and further reduction.     

Kinetic studies of Rhus vernicifera laccase. Evidence for multi-electron transfer and an oxygen intermediate in the reoxidation reaction.

1. The reoxidation of reduced Rhus vernicifera laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) by molecular oxygen has been studied by optical absorption and EPR methods. 2. The reoxidation by oxygen of the type 1 Cu+ and the two-electron acceptor is characterized by a second-order rate constant of about 5-10(6) M-1-s-1. 3. The appearance of an optical intermediate (with an absorbance maximum around 360 nm) parallels the reoxidation of type 1 Cu+ and the two-electron acceptor. It disappears in a first-order reaction with a half-time of 20 s. A similar intermediate is formed during normal turnover. 4. The type 2 Cu+ appears to be reoxidized in an intramolecular reaction with a half-time of about 20 s, suggesting a correlation between the reoxidation of this site and the disappearance of the optical intermediate. 5. The results suggest that three electrons are rapidly transferred to oxygen leading to the formation of an enzyme-bound oxygen intermediate.     

Identification of phyllostine as an intermediate of the patulin pathway in Penicillium urticae.

A patulin negative mutant (J1) of Penicillium urticae (NRRL 2159A) was found to accumulate large quantities (greater than 128 mg/L culture) of a reactive, photosensitive compound, which was isolated and identified as (-)-phyllostine (5,6-epoxygentisylquinone). This epoxyquinone possessed an antibiotic activity against Bacillus subtilis which was approximately 80% of that exhibited by patulin. In separate in vivo feeding experiments, [2-14C]acetate and [G-3H]gentisaldehyde were readily incorporated into phyllostine by mutant J1 and [14C]phyllostine was incorporated into patulin by the parent strain (NRRL 2159A). When fed to a washed-cell suspension of a second patulin negative mutant (J2) which produced gentisaldehyde but not phyllostine, unlabeled phyllostine was efficiently converted to patulin in yields of 33, 56, and 92% after 30 min, 1 and 5 h, respectively. The role of phyllostine as an intermediate of a new post-gentisaldehyde portion of the patulin biosynthetic pathway is discussed.     

C2-alpha-lactylthiamin diphosphate is an intermediate on the pathway of thiamin diphosphate-dependent pyruvate decarboxylation. Evidence on enzymes and models

Thiamin diphosphate (ThDP)-dependent decarboxylations are usually assumed to proceed by a series of covalent intermediates, the first one being the C2-trimethylthiazolium adduct with pyruvate, C2-alpha-lactylthiamin diphosphate (LThDP). Herein is addressed whether such an intermediate is kinetically competent with the enzymatic turnover numbers. In model studies it is shown that the first-order rate constant for decarboxylation can indeed exceed 50 s(-1) in tetrahydrofuran as solvent, approximately 10(3) times faster than achieved in previous model systems. When racemic LThDP was exposed to the E91D yeast pyruvate decarboxylase variant, or to the E1 subunit of the pyruvate dehydrogenase complex (PDHc-E1) from Escherichia coli, it was partitioned between reversion to pyruvate and decarboxylation. Under steady-state conditions, the rate of these reactions is severely limited by the release of ThDP from the enzyme. Under pre-steady-state conditions, the rate constant for decarboxylation on exposure of LThDP to the E1 subunit of the pyruvate dehydrogenase complex was 0.4 s(-1), still more than a 100-fold slower than the turnover number. Because these experiments include binding, decarboxylation, and oxidation (for detection purposes), this is a lower limit on the rate constant for decarboxylation. The reasons for this slow reaction most likely include a slow conformational change of the free LThDP to the V conformation enforced by the enzyme. Between the results from model studies and those from the two enzymes, it is proposed that LThDP is indeed on the decarboxylation pathway of the two enzymes studied, and once LThDP is bound the protein needs to provide little assistance other than a low polarity environment.     

15N labeling of oligodeoxynucleotides for NMR studies of DNA-ligand interactions.

The amino protons of 15N-labeled DNA were studied as a possible structural probe in NMR investigations of the interaction of DNA with various ligands. Since the imino protons are located in the center of the double helix, and variations of their chemical shift values are difficult to interpret in terms of structural changes, these probes are not very useful. Instead, amino protons are located in the major or minor groove of the DNA and are often directly involved in the binding of a ligand. For a selective probing 4-15NH2-2'-deoxycytidine and 6-15NH2-2'-deoxyadenosine were obtained by chemical synthesis. The labeled nucleosides were introduced in distinct positions of oligodeoxynucleotides by large-scale DNA synthesis. Direct 15N NMR and 1H-15N multiple quantum NMR were applied to detect the corresponding 15N labels or protons attached to the 15N labels. Chemical shift values for the cytidine and the adenosine amino nitrogen and proton resonances of a symmetric 18 base pair lac operator sequence are reported.     

Mechanism of pyruvate-uridine diphospho-N-acetylglucosamine transferase. Evidence for an enzyme-enolpyruvate intermediate.

The enzyme, phosphoenolpyruvate:uridine-5-diphospho-N-acetyl-2-amino-2-deoxyglucose-3-enolpyruvyltransferase, which catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridine diphospho-N-acetylglucosamine with the liberation of Pi, was found to form a covalent intermediate with the enolpyruvate moiety. Radioactivity from [1-14-C]phosphoenolpyruvate in the forward reaction and from UDP-GlNAc-[1-14-C]enolpyruvate in the reverse reaction was incorporated into the enzyme and remained bound to the protein after precipitation with ammonium sulfate or treatment with sodium dodecyl sulfate and heat. This incorporation from UDP-GlcNAc-[1-14-C]enolpyruvate took place in the absence of Pi. When [32-P,1-14C]phosphoenolpyruvate was used, only 14-C appeared to be incorporated. In the forward reaction, the incorporation was contingent on the removal of UDP-GlcNAc from the transferase. Consistent with the formation of an enzyme-enolpyruvate intermediate, exchange of UDP-[6-3-H]GlcNAc with UDP-GlcNAc-enolpyruvate was observed in the absence of Pi. Nonstoichiometric incorporation of 3H from 3H2O into the product, UDP-GlcNAc-enolpyruvate, was observed and was shown to be due to a product isotope effect. Based on these observations, a mechanism of action for this enzyme is proposed which involves synchronous addition-elimination followed by a second addition-elimination step.     

Structural and functional studies of an intermediate on the pathway to operator binding by Escherichia coli MetJ

We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence. The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs. The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site. In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains. Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time. The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity. In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif. Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers.     

Evidence for intermediate channelling in the glycolytic pathway of permeabilized L-929 cells

L-929 cells permeabilized by dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Unlabeled 3-phosphoglycerate (PGA) added to DSP cells reduces the specific activity of lactate coming from [14C]glucose but the extent of this reduction can not be accounted for on the basis of free diffusion of PGA coming from [14C]glucose. Studies on other glycolytic intermediates, although preliminary, yield similar results. PGA also inhibits the production of lactate from glucose; however, this effect, like that of the reduction of lactate specific activity, becomes apparent only at concentrations of PGA well in excess of those considered to be physiological. We conclude that channelling of PGA, and probably other intermediates, occurs but is of the "leaky" type.     

Evidence for an intermediate in quinolinate biosynthesis in Escherichia coli.

Evidence for the formation of an unstable intermediate in the synthesis of quinolinate from aspartate and dihydroxyacetone phosphate by Escherichia coli was obtained using toluenized cells of nadA and nadB mutants of this organism and partially purified A and B proteins in dialysis and membrane cone experiments. The results of these experiments indicate that the nadB gene product forms an unstable compound from aspartate in the presence of flavine adenine dinucleotide, and that this compound is then condensed with dihydroxyacetone phosphate to form quinolinate in a reaction catalyzed by the nadA gene product.     

Conversion of tryptophan to indolacetamide. Evidence for an electronically excited intermediate

The conversion of tryptophan to indoleacetamide in the presence of pyridoxal phosphate, horseradish peroxidase and Mn²⁺ ions goes through an electronically excited intermediate. This result which was expected on the basis of similarity of the reaction to well established bioluminescent processes, is supported by: (i) photon emission (ii) correlation between total light emission and products formation (iii) formation of pyridoxoic acid.