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M F Dunn

Publications and source records attributed to M F Dunn.

At least 73 records · Page 4Linked to original sources
PubMed1990

Characterization of precrystallization aggregation of canavalin by dynamic light scattering.

The aggregation processes leading to crystallization and precipitation of canavalin have been investigated by dynamic light scattering (DLS) in photon correlation spectroscopy (PCS) mode. The sizes of aggregates formed under various conditions of pH, salt concentration, and protein concentrations were deduced from the correlation functions generated by the fluctuating intensity of light scattered by the solutions of the protein. Results obtained indicate that the barrier to crystallization of canavalin is the formation of the trimer, a species that has been characterized by x-ray crystallographic studies (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480). The dimensions of the trimer in solution are in good agreement with those obtained both from the crystal (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480) and from a low angle x-ray scattering study in solution (Plietz, P., P. Damaschun, J. J. Müller, and B. Schlener. 1983. FEBS [Fed. Eur. Biochem. Soc.] Lett. 162:43-46). Furthermore, under conditions known to lead to the formation of rhombohedral crystals of canavalin, a limiting size is reached at high concentrations of canavalin. The size measured corresponds to an aggregate of trimers making a unit rhombohedral cell consistent with x-ray crystallographic data (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480). Presumably, such aggregates are the nuclei from which crystal growth proceeds. The present study was undertaken primarily to test the potential of DLS (PCS) as a tool for rapid, routine screening to determine the ultimate fate of protein solutions (i.e., crystallization or amorphous precipitation) at an early stage, therefore eliminating the need for long-term visual observation. Achieving this goal would constitute amajor advance in the practive of protein crystallization. Delays imposed by visual observation would be considerably reduced, and a more systematic approach could be adopted to select experimental conditions.Our findings with canavalin demonstrate that DLS(PCS) is, indeed, a selective and sensitive probe of precrystallization conditions. Other advantages of this technique include the facts that it is noninvasive, nondestructive,universal, and does not require calibration.

Computer Graphics↗
PubMed1989

Spectroscopic signatures of the T to R conformational transition in the insulin hexamer.

The cobalt(II)-substituted human insulin hexamer has been shown to undergo the phenol-induced T6 to R6 structural transition in solution. The accompanying octahedral to tetrahedral change in ligand field geometry of the cobalt ions results in dramatic changes in the visible region of the electronic spectrum and thus represents a useful spectroscopic method for studying the T to R transition. Changes in the Co2+ spectral envelope show that the aqua ligand associated with each tetrahedral Co2+ center can be replaced by SCN-, CN-, OCN-, N3-, Cl-, and NO2-. 19F NMR experiments show that the binding of m-trifluorocresol stabilizes the R6 state of zinc insulin. The chemical shift and line broadening of the CF3 singlet, which occur due to binding, provide a useful probe of the T6 to R6 transition. Due to the appearance of new resonances in the aromatic region, the 500 MHz 1H NMR spectrum of the phenol-induced R6 hexamer is readily distinguishable from that of the T6 form. 1H NMR studies show that phenol induces the T6 to R6 transition, both in the (GlnB13)6(Zn2+)2 hexamer and in the metal-free GlnB13 species; we conclude that metal binding is not a prerequisite for formation of the R state in this mutant.

Insulin↗
PubMed1989

L-serine analogues form Schiff base and quinonoidal intermediates with Escherichia coli tryptophan synthase.

Substrate analogues of L-serine have been found that react with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase. Upon reaction with alpha 2 beta 2, the analogues glycine, L-histidine, L-alanine, and D-histidine form chemical intermediates derived from reaction with enzyme-bound pyridoxal 5'-phosphate with characteristic UV-visible spectral bands. The spectra of the products of the glycine, L-histidine, and L-alanine reactions with alpha 2 beta 2 contain contributions from the external aldimine, the quinonoid species, and other intermediates along the catalytic pathway. Just as previously reported for the reaction of L-serine with beta 2 [Goldberg, M. E., York, S., & Stryer, L. (1968) Biochemistry 7, 3662-3667], the reactions of glycine, L-histidine, and L-alanine with the beta 2 form of tryptophan synthase yield spectra with no contributions from catalytic intermediates beyond the external aldimine. The kinetics of intermediate formation and comparisons of the time courses for the exchange of alpha-1H for solvent 2H catalyzed by alpha 2 beta 2 or beta 2 were found to be consistent with these assignments. Intermediates further along the tryptophan synthase catalytic pathway are stabilized to a greater degree in the alpha 2 beta 2 complex than in the beta 2 species alone. This observation strongly suggests that the association of alpha and beta subunits to form the native alpha 2 beta 2 species lowers the activation energies for the interconversion of the external aldimine with chemical species further along the catalytic path.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

Comparison of solution structural flexibility and zinc binding domains for insulin, proinsulin, and miniproinsulin.

The chromophoric divalent metal ion chelators 4-(2-pyridylazo)resorcinol (PAR) and 2,2',2"-terpyridine (terpy) are used as kinetic and spectroscopic probes to investigate in solution the SCN- -induced conformational transformations of the insulin, proinsulin, and miniproinsulin hexamers (miniproinsulin is a proinsulin analogue wherein the C-chain is replaced by a dipeptide cross-link between Gly-A1 and Ala-B30). Herein we designate the 2Zn and 4Zn crystal forms of the hexamer as the T6 and T3R3 conformations, respectively. For all three proteins, addition of SCN- reduces the rate of sequestering and removal of zinc ion by chelator. The effect of SCN- on the rate of this process saturates at the same concentration (30 mM) known to induce the T6 to T3R3 transformation in the insulin crystal. Under both T6 and T3R3 conditions, the critical stoichiometry for high-affinity interaction between Zn2+ and each of the three proteins is shown to be 2 mol of Zn2+/mol of protein hexamer. Consequently, we confirm the finding that off-axial coordination of Zn2+ via His-B10 and His-B5 residues is of minor importance for the SCN- -induced conformation change in solution [Renscheidt, H., Strassburger, W., Glatter, U., Wollmer, A., Dodson, G. G., & Mercola, D. A. (1984) Eur. J. Biochem. 142, 7-14]. Under T6 conditions, the kinetics of the reactions between insulin, proinsulin, and miniproinsulin and a variable excess of terpy are similar and biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Application of rapid-scanning, stopped-flow spectroscopy to the characterization of intermediates formed in the reactions of L- and D-tryptophan and beta-mercaptoethanol with Escherichia coli tryptophan synthase.

The reactions of the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase with D- and L-Trp and the presteady-state reaction of L-Ser and beta-mercaptoethanol under different premixing conditions have been investigated by rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy. The reaction of alpha 2 beta 2 with L-Ser and beta-mercaptoethanol occurs in 3 detectable relaxations with rates similar to the 3 relaxations seen in the partial reaction with L-Ser and in the reaction with L-Ser and indole. The presteady-state phase of the reaction of beta-mercaptoethanol with the alpha-aminoacrylate intermediate is characterized by 2 relaxations. The RSSF spectra for this reaction show that the spectral changes that take place in these 2 phases are essentially identical. The L-Trp reaction is biphasic, and the spectral changes occurring in each phase of the reaction also are identical. The 2 new spectral bands formed (lambda max congruent to 420 nm and congruent to 476 nm) are assigned as the L-Trp external aldimine (Schiff's base) and L-Trp quinonoid intermediates, respectively. The reaction of D-Trp also is biphasic. Analysis of first and second derivatives of the RSSF spectral changes give evidence for the formation of spectral bands with lambda max of approximately 423 nm, approximately 450 nm, and approximately 478 nm. The positions and shapes of these bands suggest a D-Trp external aldimine structure (423 nm) and a quinonoidal species (450 and 478 nm). However, product studies do not support this latter assignment. The behavior of the D- and L-Trp reactions and the reaction of beta-mercaptoethanol with the alpha-aminoacrylate strongly indicate the pre-existence of 2 slowly equilibrating forms of the internal aldimine and of the alpha-aminoacrylate.

Escherichia coli↗
PubMed1989

Exopolysaccharides Produced by Phytopathogenic Pseudomonas syringae Pathovars in Infected Leaves of Susceptible Hosts.

Bacterial exopolysaccharide (EPS) was extracted from infected leaves of several host plants inoculated with phytopathogenic strains of Pseudomonas syringae pathovars. Extraction was by a facilitated diffusion procedure or by collection of intercellular fluid using a centrifugation method. The extracted EPS was purified and characterized. All bacterial pathogens which induced watersoaked lesions on their host leaves, a characteristic of most members of this bacterial group, were found to produce alginic acid (a polymer consisting of varying ratios of mannuronic and guluronic acids). Only trace amounts of bacterial EPS could be isolated from leaves inoculated with a pathovar (pv. syringae) which does not induce the formation of lesions with a watersoaked appearance. Guluronic acid was either present in very low amounts or absent in the alginic acid preparations. All bacterial alginates were acetylated (7-11%). Levan (a fructan) was apparently not produced as an EPS in vivo by any of the pathogens tested.

Journal Article↗
PubMed1989

Characterization of exopolysaccharides produced by plant-associated fluorescent pseudomonads.

A total of 214 strains of plant-associated fluorescent pseudomonads were screened for the ability to produce the acidic exopolysaccharide (EPS) alginate on various solid media. The fluorescent pseudomonads studied were saprophytic, saprophytic with known biocontrol potential, or plant pathogenic. Approximately 10% of these strains exhibited mucoid growth under the conditions used. The EPSs produced by 20 strains were isolated, purified, and characterized. Of the 20 strains examined, 6 produced acetylated alginate as an acidic EPS. These strains included a Pseudomonas aeruginosa strain reported to cause a dry rot of onion, a strain of P. viridiflava with soft-rotting ability, and four strains of P. fluorescens. However, 12 strains of P. fluorescens produced a novel acidic EPS (marginalan) composed of glucose and galactose (1:1 molar ratio) substituted with pyruvate and succinate. Three of these strains were soft-rotting agents. Two additional soft-rotting strains of P. fluorescens produced a third acidic novel EPS composed of rhamnose, mannose, and glucose (1:1:1 molar ratio) substituted with pyruvate and acetate. When sucrose was present as the primary carbon source, certain strains produced the neutral polymer levan (a fructan) rather than an acidic EPS. Levan was produced by most strains capable of synthesizing alginate or the novel acidic EPS containing rhamnose, mannose, and glucose but not by strains capable of marginalan production. It is now evident that the group of bacteria belonging to the fluorescent pseudomonads is capable of elaborating a diverse array of acidic EPSs rather than solely alginate.

Journal Article↗
PubMed1988

Detection and identification of transient intermediates in the reactions of tryptophan synthase with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Evidence for a tetrahedral (gem-diamine) intermediate.

The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy. The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively). Rate constants determined by SWSF were either well resolved (i.e., k1[DHT], k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1[OXA], k-1 greater than or equal to k2, k-2 greater than k3, k-3). The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species. Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system. The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates.

Alanine↗
PubMed1988

The binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase. A probe for the open-enzyme conformation.

We have studied the binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)-LADH]. The dissociation constant is a factor of 6500 smaller than in the native enzyme. Spectral evidence is given which shows that 1,10-phenanthroline does not remove the catalytic Co(II) ion and that binding of 1,10-phenanthroline renders the catalytic metal ion pentacoordinate. The maximum limiting rate constant for the association of 1,10-phenanthroline to Co(II)-LADH is about 60 s-1. This is about a third of the value (169 s-1) determined for native horse-liver alcohol dehydrogenase, Zn(II)LADH [Frolich et al. (1978) Arch. Biochem. Biophys. 189, 471-480]. For cadmium(II)-substituted horse-liver alcohol dehydrogenase, [Cd(II)LADH] the maximum limiting rate constant for association of 1,10-phenanthroline increased to 590 s-1. These findings demonstrate that the rate-limiting step is strongly dependent on the chemical nature of the catalytic metal ion and its immediate environment. 1,10-Phenanthroline is shown to bind to the Co(II)-LADH.NAD+ complex in the open conformation. The maximum limiting rate constant remains unchanged in the presence of NAD+. The data have been used to derive a kinetic scheme for the formation of ternary complexes including NAD+ that involves a slow intermediary step.

Alcohol Dehydrogenase↗
PubMed1988

Stereoelectronic control of bond formation in Escherichia coli tryptophan synthase: substrate specificity and enzymatic synthesis of the novel amino acid dihydroisotryptophan.

The reactions of the indole analogues indoline and aniline with the Escherichia coli tryptophan synthase alpha-aminoacrylate Schiff base intermediate have been characterized by UV-visible and 1H NMR absorption spectroscopy and compared with the interactions of indole and the potent inhibitor benzimidazole. Indole, via the enamine functionality of the pyrrole ring, reacts with the alpha-aminoacrylate intermediate, forming a transient quinonoid species with lambda max 476 nm as the new C-C bond is synthesized. Conversion of this quinonoid to L-tryptophan is the rate-limiting step in catalysis [Lane, A., & Kirschner, K. (1981) Eur. J. Biochem. 120, 379-398]. Both aniline and indoline undergo rapid N-C bond formation with the alpha-aminoacrylate to form quinonoid intermediates; benzimidazole binds rapidly and tightly to the alpha-aminoacrylate but does not undergo covalent bond formation. The indoline and aniline quinonoids (lambda max 464 and 466 nm, respectively) are formed via nucleophilic attack on the electrophilic C-beta of the alpha-aminoacrylate. The indoline quinonoid decays slowly, yielding a novel, new amino acid, dihydroisotryptophan. The aniline quinonoid is quasi-stable, and no new amino acid product was detected. We conclude that nucleophilic attack requires the precise alignment of bonding orbitals between nucleophile and the alpha-aminoacrylate intermediate. The constraints imposed by the geometry of the indole subsite force the aromatic rings of indoline, aniline, and benzimidazole to bind in the same plane as indole; thus nucleophilic attack occurs with the N-1 atoms of indoline and aniline.(ABSTRACT TRUNCATED AT 250 WORDS)

Aniline Compounds↗
PubMed1988

Insulin-metal ion interactions: the binding of divalent cations to insulin hexamers and tetramers and the assembly of insulin hexamers.

An insulin hexamer containing one B10-bound Co(III) ion and one unoccupied B10 site has been synthesized. The properties of the monosubstituted hexamer show that occupancy of only one B10 site by Co3+ is sufficient to stabilize the hexameric form under the conditions of pH and concentration used in these studies. The experimentally determined, second-order rate constants for the binding of Zn2+ and Co2+ to the unoccupied B10 site are consistent with literature rate constants for the rate of association of these divalent metal ions with similar small molecule ligands. These findings indicate that the rate-limiting steps for Zn2+ and Co2+ binding involve the removal of the first aqua ligand. The rate constant for the binding of Cd2+ is significantly lower than the literature values for small molecule chelators, which suggests that some other protein-related process is rate-limiting for Cd2+ binding to the unoccupied, preformed B10 site. The kinetics of the assembly of insulin in the presence of limiting metal ion provides strong evidence indicating that the B13 site of the tetramer species can bind Zn2+, Cd2+, or Ca2+ prior to hexamer formation and that such binding assists hexamer formation. Both the tetramer and the hexamer B13 sites were found to exhibit similar affinities for Zn2+ and Cd2+ (Kd congruent to 9 microM), whereas the tetramer B13 sites bind Ca2+ much more weakly (Kd congruent to 1 mM for tetramer vs 83 microM for hexamer). The second-order rate constants estimated for the association of Zn2+ and Cd2+ to the tetrameric site indicate that the loss of the first inner-sphere aqua ligand is the rate-limiting step for binding.

Binding Sites↗
PubMed1988

1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change.

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.

Animals↗
PubMed1987

Characterization of a transient intermediate formed in the liver alcohol dehydrogenase catalyzed reduction of 3-hydroxy-4-nitrobenzaldehyde.

The compounds 3-hydroxy-4-nitrobenzaldehyde and 3-hydroxy-4-nitrobenzyl alcohol are introduced as new chromophoric substrates for probing the catalytic mechanism of horse liver alcohol dehydrogenase (LADH). Ionization of the phenolic hydroxyl group shifts the spectrum of the aldehyde from 360 to 433 nm (pKa = 6.0), whereas the spectrum of the alcohol shifts from 350 to 417 nm (pKa = 6.9). Rapid-scanning, stopped-flow (RSSF) studies at alkaline pH show that the LADH-catalyzed interconversion of these compounds occurs via the formation of an enzyme-bound intermediate with a blue-shifted spectrum. When reaction is limited to a single turnover of enzyme sites, the formation and decay of the intermediate when aldehyde reacts with enzyme-bound reduced nicotinamide adenine dinucleotide E(NADH) are characterized by two relaxations (lambda f approximately equal to 3 lambda s). Detailed stopped-flow kinetic studies were carried out to investigate the disappearance of aldehyde and NADH, the formation and decay of the intermediate, the displacement of Auramine O by substrate, and 2H kinetic isotope effects. It was found that NADH oxidation takes place at the rate of the slower relaxation (lambda s); when NADD is substituted for NADH, lambda s is subject to a small primary isotope effect (lambda Hs/lambda Ds = 2.0); and the events that occur in lambda s precede lambda f. These findings identify the intermediate as a ternary complex containing bound oxidized nicotinamide adenine dinucleotide (NAD+) and some form of 3-hydroxy-4-nitrobenzyl alcohol. The blue-shifted spectrum of the intermediate strongly implies a structure wherein the phenolic hydroxyl is neutral. When constrained to a mechanism that assumes only the neutral phenolic form of the substrate binds and reacts and that the intermediate is an E(NAD+, product) complex, computer simulations yield RSSF and single-wavelength time courses that are qualitatively and semiquantitatively consistent with the experimental data. We conclude that the LADH substrate site can be divided into two subsites: a highly polar, electropositive subsite in the vicinity of the active-site zinc and, just a few angstroms away, a rather nonpolar region. The polar subsite promotes formation of the two interconverting reactive ternary complexes. The nonpolar region is the binding site for the hydrocarbon-like side chains of substrates and in the case of 3-hydroxy-4-nitrobenzaldehyde conveys specificity for the neutral form of the phenolic group.

Alcohol Dehydrogenase↗
PubMed1987

Active-site cobalt(II)-substituted horse liver alcohol dehydrogenase: characterization of intermediates in the oxidation and reduction processes as a function of pH.

Substitution of Co(II) for the catalytic site Zn(II) of horse liver alcohol dehydrogenase (LADH) yields an active enzyme derivative, CoIIE, with characteristic Co(II) charge-transfer and d-d electronic transitions that are sensitive to the events which take place during catalysis [Koerber, S. C., MacGibbon, A. K. H., Dietrich, H., Zeppezauer, M., & Dunn, M. F. (1983) Biochemistry 22, 3424-3431]. In this study, UV-visible spectroscopy and rapid-scanning stopped-flow (RSSF) kinetic methods are used to detect and identify intermediates in the LADH catalytic mechanism. In the presence of the inhibitor isobutyramide, the pre-steady-state phase of alcohol (RCH2OH) oxidation at pH above 7 is characterized by the formation and decay of an intermediate with lambda max = 570, 640, and 672 nm for both aromatic and aliphatic alcohols (benzyl alcohol, p-nitrobenzyl alcohol, anisyl alcohol, ethanol, and methanol). By comparison with the spectrum of the stable ternary complex formed with oxidized nicotinamide adenine dinucleotide (NAD+) and 2,2',2''-trifluoroethoxide ion (TFE-), CoIIE(NAD+, TFE-), the intermediate which forms is proposed to be the alkoxide ion (RCH2O-) complex, CoIIE(NAD+, RCH2O-). The timing of reduced nicotinamide adenine dinucleotide (NADH) formation indicates that intermediate decay is limited by the interconversion of ternary complexes, i.e., CoIIE(NAD+, RCH2O-) in equilibrium CoIIE(NADH, RCHO). From competition experiments, we infer that, at pH values below 5, NAD+ and alcohol form a CoIIE(NAD+, RCH2OH) ternary complex. RSSF studies carried out as a function of pH indicate that the apparent pKa values for the ionization of alcohol within the ternary complex, i.e., CoIIE(NAD+, RCH2OH) in equilibrium CoIIE(NAD+, RCH2O-) + H+, fall in the range 5-7.5. Using pyrazole as the dead-end inhibitor, we find that the single-turnover time courses for the reduction of benzaldehyde, p-nitrobenzaldehyde, anisaldehyde, and acetaldehyde at pH above 7 all show evidence for the formation and decay of an intermediate. Via spectral comparisons with CoIIE-(NAD+, TFE-) and with the intermediate formed during alcohol oxidation, we identify the intermediate as the same CoIIE(NAD+, RCH2O-) ternary complex detected during alcohol oxidation.

Alcohol Dehydrogenase↗
PubMed1987

Effects of calcium ion on ternary complexes formed between 4-(2-pyridylazo)resorcinol and the two-zinc insulin hexamer.

As a means for probing the microenvironment of zinc in the insulin hexamer and to investigate the effects of calcium ion on the assembly and the structure of the two-zinc insulin hexamer, the thermodynamics and kinetics of the reaction between the chromophoric divalent metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) and zinc-insulin have been investigated over a wide range of conditions. For [PAR]0 much greater than [Zn2+]0 and [Zn2+]/[In] less than or equal to 0.33, the reaction leads to the sequestering and ultimate removal of all of the insulin-bound Zn2+; for [Zn2+]0 much greater than [PAR]0, two stable ternary complexes are formed where Zn2+ has ligands derived from PAR as well as from hexameric insulin. For [Zn2+]/[In] ratios below 0.33, the equilibrium distribution between the two ternary complexes is dependent on the [Zn2+]/[In] ratio. One of the complexes is assigned to the monoanion of PAR coordinated to Zn2+ that resides in a His-B10 site. The other complex is proposed to involve the coordination of (PAR)Zn to the site formed by the alpha-NH2 group of Phe-B1 and the gamma-carboxylate ion of Glu-A17 across the dimer-dimer interface on the surface of the hexamer. With either PAR or zinc-insulin in large excess, the kinetics of the PAR optical density changes are remarkably similar and biphasic. The faster step is first order in PAR and first order in insulin-bound Zn2+ (k congruent to 3 X 10(3) M-1 s-1) and involves the formation of an intermediate in which PAR is coordinated to insulin-bound zinc at the His-B10 site.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1987

Auxin production by plant-pathogenic pseudomonads and xanthomonads.

Pathogenic strains of Xanthomonas campestris pv. glycines which cause hypertrophy of leaf cells of susceptible soybean cultivars and nonpathogenic strains which do not cause hypertrophy were compared for their ability to produce indole compounds, including the plant hormone indole-3-acetic acid (IAA) in liquid media with or without supplementation with l-tryptophan. Several additional strains of plant-pathogenic xanthomonads and pseudomonads were also tested for IAA production to determine whether in vitro production of IAA is related to the ability to induce hypertrophic growth of host tissues. Indoles present in culture filtrates were identified by thin-layer chromatography, high-performance liquid chromatography, UV spectroscopy, mass spectroscopy, and gas chromatography-mass spectrometry and were quantitated by high-performance liquid chromatography. All strains examined produced IAA when liquid media were supplemented with l-tryptophan. The highest levels of IAA were found in culture filtrates from the common bean pathogen Pseudomonas syringae pv. syringae, and this was the only bacterium tested which produced IAA without addition of tryptophan to the medium. Additional indoles identified in culture filtrates of the various strains included indole-3-lactic acid, indole-3-aldehyde, indole-3-acetamide, and N-acetyltryptophan. Pseudomonads and xanthomonads could be distinguished by the presence of N-acetyltryptophan, which was found only in xanthomonad culture filtrates.

Journal Article↗
PubMed1986

Characterization of the reaction of L-serine and indole with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy.

The pre-steady-state reaction of indole and L-serine with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase has been investigated under different premixing conditions with rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy for the spectral range 300-550 nm. When alpha 2 beta 2 was mixed with indole and L-serine, the reaction of alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3) with rate constants identical with the three relaxations seen in the partial reaction with L-serine [Drewe, W.F., Jr., & Dunn, M.F. (1985) Biochemistry 24, 3977-3987]. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to be similar to the effects observed in the reaction with serine only. The observed spectral changes and isotope effects indicate that the aldimine of L-serine and PLP and the first quinoid derived from this external aldimine are transient species that accumulate during tau 1. Conversion of these intermediates to the alpha-aminoacrylate Schiff base during tau 2 and tau 3 limits the rate of formation of the second quinoidal species (lambda max 476 nm) generated via C-C bond formation between indole and the alpha-aminoacrylate intermediate. The pre-steady-state reaction of the alpha 2 beta 2-serine mixture with indole is comprised of four relaxations (1/tau 1* greater than 1/tau 2* greater than 1/tau 3* greater than 1/tau 4*).(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗