�ber eine einfache Synthese des Lactobacillus-bulgaricus-Faktors (LBF)

Wieland, Theodor; Bokelmann, Ekkehart

doi:10.1007/bf00637824

Cited by 24 publications

(3 citation statements)

References 3 publications

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“…1 Acetyl-CoA was prepared from chiral acetates (10 pmol each; specific activity 7.95 x lo5 and 7.7 x lo6 counts x min-l x pmol-l for R-and S-acetate, respectively) and coenzyme A (11 pmol each) by the method of Wieland and Bokelmann [9] in 60-65°/0 yield (determined with si-citrate synthase [lo]). Both specimens of citrate were treated likewise for synthesis of 3R-citrates as described subsequently.…”

Section: (2r3r)-[2-ahl2-3h]citrate and (2s3r)-[2-3hl]-mentioning

confidence: 99%

Stereochemistry of the re‐Citrate‐Synthase Reaction

Wunderwald¹,

Lenz²,

Buschmeier³

et al. 1971

European Journal of Biochemistry

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1. R-Acetate and S-acetate, as the coenzyme-A esters, were converted into citrates on recitrate synthase.2. The citrate were assayed for chirality a t the 2-position (a) by incubation with aconitate hydratase (aconitase) and (b) by cleavage to malate by citrate lyase combined with malate dehydrogenase, followed by incubation of the isolated malate with fumarate hydratase ( fumarase).3. I n each case, the citrate (or malate) derived from R-acetate retained most of its tritium, and the citrate (or malate) derived from S-acetate lost most of its tritium, on incubation with the h ydratase .4. It was concluded that if a normal intramolecular deuterium-isotope effect is operative, the condensation of oxaloacetate with acetyl-coenzyme A on re-citrate synthase proceeds with inversion of configuration at the methyl group. re-Citrate synthase (for nomenclature, see [14]), the fourth-known enzyme involved in citrate metabolism, was discovered by Gottschalk and Barker in extracts of anaerobic microorganisms [I]. Contrary to the action of the si-citrate lyases described in the preceding papers, this enzyme attaches the acetate unit at the re-side [2] of oxaloacetate, producing a citrate which carries the acetate derived group in pro-R position [I]. This is attacked by aconitate hydratase to yield cis-aconitate by a reversible trans-elimination of water formed from the hydroxyl group of citrate and the pro-R-hydrogen of the pro-R-acetate chain [3]. The stereochemistry of C-C bond formation on synthesis of citrate from acetylCoA and oxaloacetate consequently can be elucidated by (a) application of chiral acetates for this synthesis, and (b) use of aconitate hydratase for analysis of the product citrate.If a normal isotopic effect is operative on the re-synthase the tritium-containing products formed from R-acetate with inversion of configuration will consist of (2S,3R)-[2-2H,,2-3Hl]citrate preponderating over (2R,3R)-[2-3H,]citrate in a ratio equal to the intramolecular JCHIJCD for the re-synthase. This mixture on incubation with aconitate hydratase will retain more than 50°/, of its radioactive label. Retention of configuration would yield a mixture of (2R,3R)-[2-aHl,2-3H,]citrate and (2S,3R)-[2-3Hl]-citrate where the former outweighs the latter. This mixture on incubation with aconitate hydratase will lose more than 50°/, of the tritium label into the medium. Hence aconitate hydratase for analysis of stereospecifically labelled R-citrates provides the same analytical tool as does fumarase in case of the malates [4,5].Another approach to reveal the stereochemistry of the re-synthase makes use of the enzymes citrate lyase and malate dehydrogenase. Here the acetatederived group of R-citrate in the presence of NADH is converted to malate via oxaloacetate and malate is subjected to the usual analysis with fumarase. Melate dehydrogenase transfers hydrogen from NADH to the re side of oxaloacetate and fumarase, like aconitate hydratase, catalyzes a trans-elimination of water [3]. The hydrogen eliminated in the formation of water in ...

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Section: (2r3r)-[2-ahl2-3h]citrate and (2s3r)-[2-3hl]-mentioning

confidence: 99%

Stereochemistry of the re‐Citrate‐Synthase Reaction

Wunderwald¹,

Lenz²,

Buschmeier³

et al. 1971

European Journal of Biochemistry

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“…Bromacetyl-Pantethein erhielten wir durch Umsetzung von Pantethein mit dem gemischten Anhydrid aus Bromessigsaure und Kohlens&ureiithylester [45,38] ; Gehaltsbestimmung mit der Hydroxamsaure-Methode [46].…”

Section: S-acetacetyl-n-(n-acetacetyl-b-alanyl) -Cystearninunclassified

Reinigung und Kristallisation der Thiolase, Untersuchungen zum Wirkungsmechanismus

Gehring

Riepertinger

Lynen

1968

European Journal of Biochemistry

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Thiolase was purified 600-700 fold from a crude extract of pig heart by use of differential adsorption on Al( OH), gel, ammonium sulfate fractionation and ion exchange chromatography on DEAE-Sephadex, CM-Sephadex and phosphorylated cellulose. The enzyme preparation is homogeneous by the following criteria : sedimentation analysis and electrophoresis on polyacrylamide gel and on cellulose acetate membranes. The isoelectric point of the protein was found to be approximately pH 7.2-7.3.The enzyme crystallized from ammonium sulfate solutions as thin plates of irregular shape. Crystalline thiolase stored for 18 months a t -10" did not show any loss of activity.The thiolytic activity and the acyl transferase activity of the enzyme could be demonstrated separately.The thiolytic activity was demonstrated by the specific incorporation of 14C from [1J4C]-acetyl-CoA into the carboxyl position of unlabelled acetoacetyl-CoA on incubation with the enzyme. The acyl transferase activity was studied using either [l-14C]acetyl-CoA and pantethein as substrates or 1%-labelled CoA and acyl-CoA derivatives of various chain length. A remarkable specificity was found for the transfer of the acetyl group.Thiolase activity and acyl transferase activity of the enzyme were diminished with the same kinetics during heat treatment or incubation with N-ethylmaleimide, p-chloromercuribenzoate or iodoacetamide. This indicates that both activities belong to the same protein. Inhibition of thiolase and acyltransferase activities by iodoacetamide (5 x M) could be prevented by preincubation of the enzyme with acetoacetyl-CoA or acetyl-CoA. The acyl transfer is the rate determining step when the thiolase reaction operates in the direction of chain cleavage.Incubation of thiolase with acetyl-CoA results in binding of acetyl groups to the protein. The uptake of acetyl groups is dependent upon the specific activity of the enzyme and corresponds to 3 acetyl residues per molecule of protein (molecular weight 170,000) for the highest enzymatic activity obtained. Both incorporation of acetyl groups into the enzyme and thiolase activity are inhibited to the same extent by iodoacetamide.The incorporation of radioactivity into the enzyme during incubation with iodo-[ 1 -14C]-acetamide can be divided into two kinetically different reactions: a fast incorporation with accompanying loss of enzymatic activity and a much slower process which continues after complete inactivation. From the initial rate of incorporation, it was calculated that incorporation of 3.1 carbamoylmethyl residues per molecule of protein would completely inhibit the enzyme with the highest obtained activity. This ratio compares well with the results obtained by acetyl binding studies and indicates a minimum of 3 active sites per molecule of enzyme.Enzyme labelled with i~do-[l-~~C]acetamide was hydrolyzed with 6 N I-ICI. Approximately 95 of the radioactivity in the hydrolysate was found to coincide with S-carboxymethyl-Lcysteine on electrophoresis and paper chromatography. Repeated crystalliza...

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“…Thereafter, Wieland et al 2 ) synthesized pantetheine by the condensation of ethyl pantotheny1carbonate with cystamine hydrochloride in the presence of 2-N sodium hydroxide, Baddiley et al,a,9) King et al, 4) and Snell et al 6 ) by the condensation of pantolactone with N-!3-alanyl-!3-mercaptoethylamine, Wittle et al,) by the condensation of methyl pantotht:nate (or pantothenoyl azide) with cystamine, or of (-)-pantolactone with N-!3-alanyl-!3-mercaptoethylamine (or the disulfide), Snell et al,7l also, by the condensation of ethyl pantothenate with cystamine (!3-mercaptoethylamine), and Funahashi et al,lOl at first, synthesized pantethine by the condensation of ethyl pantothenate with cystamine, and then obtained pantetheine by reducing it. Walton et al 6 ) condensed pantolactone with N-!3-alanyl-S-benzyl-2-thiothylamine, and then obtained pantetheine by hydrogenolysis of the benzyl group.…”

mentioning

confidence: 99%