Glykosidasen

Wallenfels, Kurt; Diekmann, H.

doi:10.1007/978-3-662-30541-6_28

Cited by 5 publications

(6 citation statements)

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“…Fractions of 9 drops each were collected. Cytochrome c was determined by the absorbance at 544 nm, haemoglobin at 420 nm, yeast alcohol dehydrogenase by the method ofVallee and Hoch [19], S-galactose dehydrogenase according to Wallenfels and Kurz [20] and pyruvate kinase by the procedure of Bucher and Pfleiderer [21]. The molecular weight of glutamate-semialdehyde dehydrogenase was calculated by the methods of Martin and h i e s [18] and Paetkau and Lardy [22].…”

Section: Sucrose-gradien T Ceri T Rifugat Ionmentioning

confidence: 99%

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Hayzer

Leisinger²

1982

Eur J Biochem

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Glutamate-semialdehyde dehydrogenase, catalysing the reduction in vivo of y-glutamyl phosphate to glutamate 5-semialdehyde in the pathway of proline biosynthesis in Escherichia coli, has been purified to homogeneity. High initial levels of the enzyme were achieved by using a multicopy ColEl-proA,B hybrid plasmid. The protein has a molecular weight of 1.89 x lo5 and consists of four identical subunits of molecular weight 4.7 x lo4 each. The pH optimum is 7.0 and the protein is stable for at least 10 min between pH 6.0-9.0 and for long periods at pH 7.0. It is rapidly inactivated at temperatures greatcr than 50 "C. The enzyme is very sensitive to inhibition byp-chloromercuribenzoate, copper and nickel ions.The synthesis of L-proline by Escherichia coli involves the reduction of L-glutamic acid to glutamate hemialdehyde, mediated by two consecutive enzymic steps. A y-glutamyl kinase (ATP : L-glutamate 5-phosphotransferase, EC 2.7.2.1 1) activates the y-carboxyl group of glutamate before its reduction to an aldehyde by NADPH-dependent glutamatesemialdehyde dehydrogenase [~-glutamate-5-semialdehyde : NADP+ oxidoreductase (phosphorylating), EC 1.2.1.41, or y-glutamyl-phosphate reductase]. Glutamate 5-semialdehyde undergoes spontaneous Schiff-base formation and cyclisation to L-1-pyrroline-5-carboxylic acid which is subsequently reduced to proline by an NADPH-dependent pyrroline-5-carboxylic acid reductase [L-proline : NAD(P) + 5-0x1-doreductase, EC 1.5.1.21. All three biosynthetic enzymes from Pseudomonas aeruginosa PA01 have been partially purified and characterized [1,2] whereas the first two of the E. coli biosynthetic enzymes have been subjected to investigation whilst in a very crude state [3,4]. Pyrroline-5-carboxylic acid reductase of E. coli, a highly active and readily measurable enzyme, has been partially purified and characterized [ 5 ] . The availability of pure samples of the first two enzymes of the pathway would enable a study to be made of the problems of the true nature of the activated intermediate in the pathway, generally assumed to by y-glutamyl phosphate, and whether there is aggregation between the kinase and glutamatesemialdehyde dehydrogenase. The function of such an enzyme complex would be to protect the glutamyl phosphate from a hostile nucleophilic and aqueous environment. To date these studies have been limited by the use of crude cell extracts but have provided suggestive evidence of the existence of an aggregate [6-81. This paper describes a method for the purification of the second enzyme in the proline biosynthetic pathway and its physical properties. MATERIALS AND METHODS Bacrerial StrainThe construction of Eschevichia coli strain CSH52/ pLC44-11 [ihi araA(lac pro)strA recA($J8Odlaci)(pLC44-1 I)] has been described by Hayzer and Leisinger [9].Large-Scale Fermentation of E. coli CSH52/pLC44-1 I A preculture of 300 ml of medium M63 [lo] plus glucose (0.5% w/v) in a 1-1 flask was grown overnight at 37°C with vigorous aeration. The entire amount was used to inocu1;lte 7 1 of the same med...

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Section: Sucrose-gradien T Ceri T Rifugat Ionmentioning

confidence: 99%

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Hayzer

Leisinger²

1982

Eur J Biochem

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“…XH Groups Kinetic measurements of the alkylation rates of cysteine SH groups were done in 0.1 M KC1 solution under a nitrogen atmosphere. The reaction rate was monitored with the Radiometer TTT la/SBR 2b automatic titrator as described earlier [8]. The reaction volume was 5.5m1, the temperature was held constant within &O.lo.…”

Section: Kinetic Nemurements Of Alkylation Rates Of Cysteinementioning

confidence: 99%

Stereospecific Alkylation with Asymmetric Reagents

Wallenfels

Eisele

1968

European Journal of Biochemistry

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1.When undergoing nucleophilic reactions with proteins, a-iodopropionic acid and its amide show greater specificity for SH groups than do iodoacetic acid and its amide.2. The kinetics and stereochemistry of the reactions of the D( +) and L( -) antipodes of a-iodopropionic acid and its amide with both cysteine and papain were investigated. The antipodes of the SH reagents reacted with these asymmetric SH compounds a t different rates. I n the reaction with papain, the L( -) antipode of the acid reacted faster than the D(+) antipode; the stereochemical preference was inverted when the amide was used, the D(+)antipode reacting faster than the L( -)antipode. 3.Arguing from the differing reactivities of the SH reagents and their antipodes and the pH dependence of the reaction rates, it has been suggested, that L( -)a-iodopropionic acid is specifically oriented onto the reaction center through the concerted attractive and repulsive actions of a cationic and an anionic group in the protein. These have been tentatively identified as a protonated imidazole residue and a carboxylate group respectively.4. Parallels between these stereospecific alkylations and the catalytic action of the enzyme are discussed.Gundlach, Stein, and Moore [1] demonstrated in their studies of the alkylation of ribonuclease with iodoacetic acid and iodoacetamide, that these "classic" sulfhydryl reagents do not react only with SH groups. Other nucleophiles present in proteins, such as amino, imidazolyl or thioether groups may also be alkylated. The extent of these side reactions depends on the reaction conditions. Our purpose in undertaking this work was to find SH reagents with higher selectivity for SH groups.Reasoning from a theory of Bunnett [a] it seemed likely, that this could be achieved by the introduction of polarizable substituents alpha to the halogen of halogen-alkyles. The possibility for the action of dispersion forces, which is introduced with the polarizable substituent, should specially favor the transition state for the reaction with the highly polarizable SH group. Steric, inductive or mesomeric effects of the a substituent would be expected to show the same influence on the reactions with all different nucleophilic groups.We tested this hypothesis using a series of derivatives of iodoacetamide with different substituents alpha to the iodine. With the methyl group as CI substituent i e . in the reaction of amino acids with a-iodopropionamide we observed an enhanced selectivity for SH groups as predicted by Bunnett's theory A further possibility for selective reactivity is contributed by a-substitution in iodoacetic acid derivatives ; the leaving group of the reagent is attached to an asymmetric carbon atom. One may expect specific interactions if the substrate itself is asymmetric since diastereomeric transition states are involved. This type of stereoselective reaction was shown for the first time by Heinrikson et al. [4,5]. They found, that the essential histidine (residue 12) of ribonuclease reacts preferentially with the D-...

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“…All analogues studied were substrates for the sialyltransferase. Activation and transfer of 9-amino-NeuAc is of considerable Galactose content was determined after acid hydrolysis of the glycoprotein (1 M HC1, 1 h, 2 h and 4 h at 100°C) using the galactose dehydrogenase assay [24].…”

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confidence: 99%

Activation and transfer of novel synthetic 9‐substituted sialic acids

Gross

Bünsch

Paulson

et al. 1987

European Journal of Biochemistry

109

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In this report several NeuAc analogues differently modified at position C-9 were tested as substrates for CMPsialic acid synthase from bovine brain: the hydroxy group at C-9 was replaced by an amino, acetamido, benzamido, hexanoylamido and azido group.The synthase was partially purified by chromatography on CDP-hexanolamine -Sepharose. CMP-glycosides synthesized were measured by analytical HPLC at 275 nm.Each NeuAc analogue was activated to the respective CMP-glycoside: K,-values varied from 0.8 mM to 4.6 mM, the K,,, for NeuAc was 1.4 mM. Thus affinity of the enzyme was influenced only moderately by chemical modification at C-9.CMP-glycosides were synthesized on a preparative scale with good yield and characterized by analytical HPLC. in addition, 500-MHz 'H-NMR data of CMP-9-amino-NeuAc and CMP-9-acetamido-NeuAc were obtained.Each CMP-activated NeuAc analogue was a suitable donor substrate for GalP 1-4GlcNAc cr2,6-sialyltransferase from rat liver. Transfer was determined by the thiobarbituric acid method and by analytical HPLC at 200 nm. The results demonstrate that synthetic, not naturally occurring, non-labelled NeuAc analogues can be incorporated into glycoprotein with high yield. Abbreviations. NeuAc, N-acetyl-D-neuraminic acid; 9-aminoNeuAc, 5-acetamido-9-amino-3,5,9-trideoxy-2-nonulosonic acid; 9-acetamido-NeuAc, 5,9-diacetamido-3,5,9-trideoxy-2-nonulosonic acid; 9-hexanoylamido-NeuAc, 5-acetamido-3,5,9-trideoxy-9-hexanoylamido-2-nonulosonic acid; 9-benzamido-NeuAc,5-acetamido-9-benzamido-3,5,9-trideoxy-2-nonulosonic acid; 9-azido-NeuAc, 5-acetamido-9-azido-3,5,9-trideoxy-2-nonulosonic acid ; 9-iodo-NeuAc, 5-acetamido-3,5,9-trideoxy-9-iodo-2-nonulosonic acid; 9-fluoroNeuAc, 5-acetamido-3,5,9-trideoxy-9-fluoro-2-nonulosonic acid; C,-NeuAc, 5-acetamido-3,5-dideoxy-2-heptulosonic acid; CMPNeuAc, cytidine-5'-monophospho-N-acetylneuraminic acid; 9-0-acetyl-NeuAc, 5-acetamido-9-O-acetyl-3,5-dideoxy-2-nonulosonic acid.Enzymes. CMPsialic acid synthase (EC 2.7.7.43); Galpl4GlcNAc a-2,6-sialyltransferase (EC 2.4.99.1); sialidase, acylneuraminylhydrolase (EC 3.2.1.18). cells may be an important biological role of sialic acids [4, 91. But many questions concerning the biological significance of this acid still remain to be answered. Further insight into these problems could be gained by studying synthetic sialic acid analogues. A substitution of natural sialic acid of glycoproteins and glycolipids with synthetic analogues would be of particular interest as the biological role of sialic acids and the correlation between structure and function of sialic acid can be investigated. In principle this could be done enzymatically by removing sialic acid with sialidase, followed by replacement with sialyltransferase and the appropriate donor substrate. In order to carry out such investigations the substrate specificity of CMP sialic acid synthase and sialyltransferase for each NeuAc analogue has to be established first.To date, only a few synthetic analogues have been used as substrate for the synthase. Although the activati...

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Glykosidasen

Cited by 5 publications

References 213 publications

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Stereospecific Alkylation with Asymmetric Reagents

Activation and transfer of novel synthetic 9‐substituted sialic acids

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