2D electron temperature diagnostic using soft x-ray imaging technique.

We have developed a two-dimensional (2D) electron temperature (T(e)) diagnostic system for thermal structure studies in a low-aspect-ratio reversed field pinch (RFP). The system consists of a soft x-ray (SXR) camera with two pin holes for two-kinds of absorber foils, combined with a high-speed camera. Two SXR images with almost the same viewing area are formed through different absorber foils on a single micro-channel plate (MCP). A 2D Te image can then be obtained by calculating the intensity ratio for each element of the images. We have succeeded in distinguishing T(e) image in quasi-single helicity (QSH) from that in multi-helicity (MH) RFP states, where the former is characterized by concentrated magnetic fluctuation spectrum and the latter, by broad spectrum of edge magnetic fluctuations.

Tangential soft-x ray imaging for three-dimensional structural studies in a reversed field pinch.

Tangential soft-x ray (SXR) imaging diagnostic has been developed and three-dimensional (3D) structure of the internal magnetic surface has been deduced by comparing the experimental and calculated two-dimensional SXR images in a reversed field pinch. The SXR imaging system, consisting of a MCP, a fluorescent plate, and an intensified charge coupled device camera, has been installed in REversed field pinch of Low-Aspect-ratio eXperiment (RELAX) machine. Major characteristics of an experimental SXR image could be reproduced by numerical calculations of the image using a single island model, suggesting a helical hot core in RELAX. The SXR imaging system could be useful for 3D structural studies when tangential and vertical simultaneous imaging systems would be installed, with appropriate numerical modeling of 3D structure of the magnetic surfaces.

Self-reversal phenomena of toroidal current by reversing the external toroidal field in helicity-driven toroidal plasmas.

In order to understand self-organization in helicity-driven systems, we have investigated the dynamics of low-aspect-ratio toroidal plasmas by decreasing the external toroidal field and reversing its sign in time. Consequently, we have discovered that the helicity-driven toroidal plasma relaxes towards the flipped state. Surprisingly, it has been observed that not only toroidal flux but also poloidal flux reverses sign spontaneously during the relaxation process. The self-reversal of the magnetic fields is attributed to the nonlinear growth of the n=1 kink instability of the central open flux.

Catalytic antibodies induced by a zwitterionic hapten.

A zwitterionic hapten 4 featuring both positively and negatively charged functional groups was designed and synthesized with the goal of generating catalytic antibodies for the hydrolysis of ester 6 and amide 7. Of the 36 monoclonal antibodies specific to BSA-4 (bovine serum albumin) that were isolated, six accelerated the hydrolysis of 6. Two catalytic antibodies with distinctively different and representative kinetic behaviors were selected for detailed kinetic studies. Whereas H8-2-6F11 showed burst kinetic behavior, which can be attributed to the formation of an acyl intermediate, H8-1-2D5 did not, but it did exhibit high multiple turnover activity. The rate of hydrolysis of 6 catalyzed by H8-1-2D5 followed Michaelis-Menten kinetics; the apparent values of the Michaelis-Menten constant Km and the catalytic constant kcat were 488 microM and 3.5 min(-1), respectively. The catalytic rate enhancement (kcat/kun) observed for H8-1-2D5 was 1.3 x 10(5), which is approximately two orders of magnitude greater than those for monofunctional haptens. Thus H8-1-2D5 compares well in catalytic activity with antibodies isolated by a related approach called heterologous immunization.

Frustrations in polymer conformation in gels and their minimization through molecular imprinting.

We report an experimental realization of a gel system in which frustrations exist and can be minimized, thus meeting two crucial criteria predicted to enable memory of conformations in polymers. The gels consist of a thermosensitive major monomer component and two minor components. One minor component is positively charged and will form complexes around negatively charged target molecules placed in solution. The complexes can be imprinted into the gel by then cross-linking the second minor component, which will form cross-links additional to those in the major polymer matrix. The complexes are destroyed and reformed upon swelling and reshrinking of the gels, showing that memorization has been achieved.

Gel catalysts that switch on and off.

We report development of a polymer gel with a catalytic activity that can be switched on and off when the solvent composition is changed. The gel consists of two species of monomers. The major component, N-isopropylacrylamide, makes the gel swell and shrink in response to a change in composition of ethanol/water mixtures. The minor component, vinylimidazole, which is capable of catalysis, is copolymerized into the gel network. The reaction rate for catalytic hydrolysis of p-nitrophenyl caprylate was small when the gel was swollen. In contrast, when the gel was shrunken, the reaction rate increased 5 times. The activity changes discontinuously as a function of solvent composition, thus the catalysis can be switched on and off by an infinitesimal change in solvent composition. The kinetics of catalysis by the gel in the shrunken state is well described by the Michaelis-Menten formula, indicating that the absorption of the substrate by the hydrophobic environment created by the N-isopropylacrylamide polymer in the shrunken gel is responsible for enhancement of catalytic activity. In the swollen state, the rate vs. active site concentration is linear, indicating that the substrate absorption is not a primary factor determining the kinetics. Catalytic activity of the gel is studied for substrates with various alkyl chain lengths; of those studied the switching effect is most pronounced for p-nitrophenyl caprylate.

Reversible molecular adsorption based on multiple-point interaction by shrinkable gels.

A general approach is presented for creating polymer gels that can recognize and capture a target molecule by multiple-point interaction and that can reversibly change their affinity to the target by more than one order of magnitude. The polymers consist of majority monomers that make the gel reversibly swell and shrink and minority monomers that constitute multiple-point adsorption centers for the target molecule. Multiple-point interaction is experimentally proven by power laws found between the affinity and the concentration of the adsorbing monomers within the gels.

Design and syntheses of three haptens to generate catalytic antibodies that cleave amide bonds with nucleophilic catalysis.

Design principles and syntheses of three haptens that were recently reported to generate amide bond cleaving catalytic antibodies are described. The hapten designs sought to induce acidic and/or basic residues in antibody binding sites via charge complementarity, and also to generate a hydrophobic binding pocket for an external phenol nucleophile. The charged yet aromatic nature of these haptens presented some unique synthetic challenges and solutions to which are described below.

Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity.

Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease-deficient E. coli UT5600 (ompT-). Induction with IPTG results in soluble PHA synthase, which is approximately 5% of the total protein. The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylapatite and Q-Sepharose and has a specific activity of 5 mumol min-1 mg-1. The molecular weight of the PHA product is approximately 10(6) Da based on PlGel chromatography and calibration using polystyrene molecular weight markers. The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms. Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA. Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization the elongation process occurs at a rate much faster than during the initiation process. A mechanistic model has been proposed for the polymerization process [Griebel, R., Smith, Z., & Merrick, J. (1968) Biochemistry 7, 3676-3681] in which two cysteines are required for catalysis. This model is based on the well-characterized enzymes involved in fatty acid biosynthesis. To test this model, several site-directed mutants of synthase, selected based on sequence conservation among synthases, have been prepared. The C459S mutant has activity approximately 90% that of the wt protein, while the C319S and C319A synthases possess < 0.01% the activity of the wt protein. CD and antibody studies suggest that the mutant proteins are properly folded. The detection of only a single essential cysteine by mutagenesis and the requirement for posttranslational modification by phosphopantetheine to provide a second thiol in many enzymes utilizing coenzyme A thiol ester substrates made us consider the possibility that posttranslational modification was required for synthase activity as well. This hypothesis was confirmed when the plasmid containing PHA synthase (pKAS4) was transformed into E. coli SJ16, requiring beta-alanine for growth. Growth of SJ16/pKAS4 on [3H]-beta-alanine followed by Coomassie staining of the protein and autoradiography revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein. These results suggest PHA synthase is posttranslationally modified by phosphopantetheine.(ABSTRACT TRUNCATED AT 400 WORDS)

Biosynthetic thiolase from Zoogloea ramigera. Mutagenesis of the putative active-site base Cys-378 to Ser-378 changes the partitioning of the acetyl S-enzyme intermediate.

The proposed active-site base Cys-378 of thiolase, responsible for deprotonation of acetyl-CoA, has been converted to a less acidic residue Ser-378 by mutagenesis. Comparison of the CD spectra and dimethyl suberimidate cross-linking experiments of the wild type, mutant Ser-378, and Gly-378 enzymes indicated that there have been no major conformational changes. The Ser-378 enzyme retains 0.1% of the Vmax of wild type in the direction of acetoacetyl-CoA thiolytic cleavage and 0.07% of the Vmax in the Claisen condensation direction. Analysis of the acetyl S-enzyme intermediate partitioning, that is capture of the acetyl enzyme by 1) the thiolate of coenzyme A relative to 2) the C-2 carbanion of acetyl-CoA, is changed to favor reaction 2 in the case of the Ser-378 mutant enzyme.

Biosynthetic thiolase from Zoogloea ramigera. Evidence for a mechanism involving Cys-378 as the active site base.

Biosynthetic thiolase from Zoogloea ramigera was inactivated with a mechanism-based inactivator, 3-pentynoyl-S-pantetheine-11-pivalate (3-pentynoyl-SPP) where K1 = 1.25 mM and kinact = 0.26 min-1, 2,3-pentadienoyl-SPP obtained from nonenzymatic rearrangement of 3-pentynoyl-SPP where K1 = 1.54 mM and kinact = 1.9 min-1 and an affinity labeling reagent, acryl-SPP. The results obtained with the alkynoyl and allenoyl inactivators are taken as evidence that thiolase from Z. ramigera is able to catalyze proton abstraction uncoupled from carbon-carbon bond formation. The inactivator, 3-pentynoyl-SPP and the affinity labeling reagent, acryl-SPP, trap the same active site cysteine residue, Cys-378. To assess if Cys-378 is the active site residue involved in deprotonation of the second molecule of acetyl-CoA, a Gly-378 mutant enzyme was studied. In the thiolysis direction the Gly-378 mutant was more than 50,000-fold slower than wild type and over 100,000-fold slower in the condensation direction. However, the mutant enzyme was still capable of forming the acetyl-enzyme intermediate and incorporated 0.81 equivalents of 14C-label after incubation with [14C]Ac-CoA for 60 min. The reversible exchange of 32P-label from [32P]CoASH into Ac-CoA, catalyzed by the Gly-378 mutant enzyme, proceeded with a Vmax (exchange) 8,000-fold less than the wild type enzyme but at least 10-fold faster than the overall condensation reaction. These data provide evidence that Cys-378 is the active site base.

Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes.

Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys89 by acetylation with [14C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degrees C. Cys89 has been converted to the alternate nucleophile Ser89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser89 enzyme retains 1% of the Vmax of the Cys89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the Vmax in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [14C]acetyl-CoA and isolation of the labeled Ser89-containing tryptic peptide. Comparisons of the Cys89 and Ser89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [32P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA.

The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera. Characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli.

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3-hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/mg). The enzyme is an alpha 4 homotetramer of four 25-kDa subunits. It has a Km of 2 microM and a kcat/Km of 1.8 X 10(8) M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyl-CoA above 10 microM. K is 10(-10) M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase.

Biosynthetic thiolase from zoogloea ramigera. I. Preliminary characterization and analysis of proton transfer reaction.

The biosynthetic thiolase, from Zoogloea ramigera, involved in generation of acetoacetyl-CoA for poly-beta-hydroxybutyrate synthesis, has been prepared pure in quantity for initial structural characterization of this homotetrameric enzyme. Edman degradation provided the sequence of the NH2 terminal 25 residues and an active site cysteine-containing nonapeptide labeled on stoichiometric inactivation by iodoacetamide. Both sequences were used to align the encoding DNA sequence of the cloned gene as described in an accompanying paper. Synthetic analogs of acetoacetyl-S-CoA, modified in the CoA moiety, were prepared and tested, and acetoacetyl-S-pantetheine 11-pivalate 1 was shown to have a kcat/Km of 6.4 X 10(6) M-1 s-1, comparable to the kcat/Km of 2 X 10(7) M-1 s-1 for acetoacetyl-S-CoA. The pantetheine pivalate group facilitates nonaqueous synthetic manipulations and may be generally useful as a CoA replacement. We have also prepared the carba analog of 1, with CH2 replacing S, to yield a beta-diketone analog 10 of acetoacetyl-S-CoA and the corresponding methyl ketone analog 9 of acetyl-S-CoA. These analogs have been used to prove the ability of Z. ramigera thiolase to catalyze proton abstraction from the C-2 methyl group of the acetyl portion of substrate in a transition state separate from C-C bond formation. NMR studies in D2O show exchange only when condensation is possible. Further studies with [2-3H]acetyl-CoA show there is neither pre-equilibrium washout nor detectable kH/kT expressed in turnover and provide no evidence for a discrete acetyl-CoA C-2 carbanion or a nonconcerted reaction.

Biosynthetic thiolase from Zoogloea ramigera. II. Inactivation with haloacetyl CoA analogs.

The thiolase involved in biosynthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera generates an acetyl-enzyme species during catalysis. Up to 0.86 [14C] acetyl eq/subunit of this homotetrameric enzyme is accumulated by acid precipitation in the presence of [14C]acetyl-CoA. Gel filtration of the same solutions produced only 7% acetyl-enzyme suggesting hydrolytic lability of the acetyl-enzyme during the 10-min isolation at 4 degrees C. In an effort to identify active site residues which may function as basic groups to deprotonate at C-2 of acetyl-CoA to generate the required nucleophilic equivalent in carbon-carbon bond formation, we have prepared and tested haloacetyl-thioesters, oxoesters, and amides in the panthetheine pivalate series (Davis, J. T., Moore, R. N., Imperiali, B., Pratt, A. J., Kobayashi, K., Masamune, S., Sinskey, A. J., and Walsh, C. T. (1987) J. Biol. Chem. 262, 82-89). The [14C]bromoacetyl-oxoester alkylatively inactivates thiolase irreversibly with stoichiometric incorporation of four labels/tetramer. Determination of amino acid composition of the radiolabeled tryptic peptide indicated trapping of Cys-89 (Peoples, O. P., Masamune, S., Walsh, C. T., and Sinskey, A. J. (1987) J. Biol. Chem. 262, 97-102), the same residue modified by iodoacetamide. When the bromoacetyl-thioester was used, inactivation was pH-dependent. The data are consistent with the competition of two processes, acylation, and alkylation. Direct (rather than secondary) alkylation of thiolase by the inactivator accounts for the significant 14C incorporation into thiolase with the thioester labeled with [14C] in the pantetheine pivalate moiety. It appears likely that the haloacetyl analogs described herein should be generally useful for affinity labeling other enzymes using acetyl-CoA as a substrate.

Biosynthetic thiolase from Zoogloea ramigera. III. Isolation and characterization of the structural gene.

The gene coding for the biosynthetic thiolase from Zoogloea ramigera has been isolated by using antibody screening methods to detect its expression in Escherichia coli under the transcriptional control of the lac promoter. We have located and determined the nucleotide sequence of the gene. The structural gene is 1173 nucleotides long and codes for a polypeptide of 391 amino acids; 282 nucleotides 5' and 58 nucleotides 3' to the coding sequence are also reported. By comparing the amino acid sequence data predicted from the gene with data determined experimentally, we have derived the complete primary structure of thiolase. A catalytically essential cysteine is located at residue 89. The DNA sequence presented has a very high G/C content, 66.2%, typical of the Z. ramigera genome. In the coding region, this increases to 68.2% and is strongly reflected in the codon usage which demonstrates a strong preference for G or C in the third position. Examination of the 5'-flanking sequence establishes that the NH2-terminal methionine is specified by an ATG codon, 7 nucleotides downstream from a Shine-Dalgarno sequence.

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T1 has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(-)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(-)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.

Total synthesis of the L-hexoses.

Enantiomerically pure polyhydroxylated natural products are synthesized by using a reiterative two-carbon extension cycle consisting of four steps. The generality and efficiency of this methodology are demonstrated in the total synthesis of all eight L-hexoses.