Enzymic Generation of Chiral Acetates. A Quantitative Evaluation of Their Configurational Assay

Lenz, Helmut; Eggerer, Hermann

doi:10.1111/j.1432-1033.1976.tb10410.x

Cited by 33 publications

(33 citation statements)

References 16 publications

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“…The chirality of the asymmetric methyl groups was determined by the use of two enzymes catalysing reactions of known stereochemical course. (8) at C-2 of acetate [18,19,22]. Fumarase catalyses the equilibration of the C-3 pro-R hydrogen of malate with the protons of the medium [20,21] The simplest mechanism to achieve this inversion is an S N~ type of displacement of the amino group by hydride.…”

Section: Discussionmentioning

confidence: 99%

Mechanistic and Stereochemical Studies on the Glycine Reductase of Clostridium sticklandii

Barnard

Akhtar

1979

European Journal of Biochemistry

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Clostridial glycine reductase multienzyme complex which catalyses the reaction :Glycine + ADP + Pi + 2H -+ Acetate + ATP + NH3 was solubilised and fractionated essentially according to the method of Stadtman [T. C. Stadtman (1970) Methods Enzymol. I7A, 956 -9661 into two components : protein A and 'glycine reductase' fraction. A reconstituted system obtained by combining the two components in the presence of dithiothreitol catalysed the conversion of glycine into acetate concomitant with the phosphorylation of ADP to ATP. Using the reconstitued system, in which the unwanted enzymic activity catalyzing an exchange of the ct hydrogen atoms of glycine with the protons of the medium had been greatly reduced, it was found that the conversion of (2RS)-[2-14C, 2-3H1]glycine (3H/'4C = 7.16) into acetate (3H/14C = 7.03) was attended by the retention of both the C-2 hydrogen atoms of glycine. Conversion of (2S)-[2-2H1, 2-3Hl]glycine and (2R)-[2-2H1, 2-3Hl]glycine by the reconstituted system gave (2S)-acetate and (2R)-acetate respectively showing that the reductive deamination of glycine occurs through an inversion of configuration. The cumulative information available on the glycine reductase reaction is embodied in a hypothetical mechanism of action for the enzyme.

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Section: Discussionmentioning

confidence: 99%

Mechanistic and Stereochemical Studies on the Glycine Reductase of Clostridium sticklandii

Barnard

Akhtar

1979

European Journal of Biochemistry

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“…As a further refinement to keep oxaloacetate as short-lived as possible, limiting amounts of malate dehydrogenase relative to oxalacetase were used. Both variations were applied in experiments where fast turnover was important [5].…”

Section: Direct Optical Determination Jrom Oxaloacetate Absorption (Amentioning

confidence: 99%

“…Assay 2 was the most convenient and used routinely for all determinations of oxalacetase activity during enzyme purification and determination of kinetic constants. Assay 3 could only be used with purified enzyme preparations; its main application was restricted to chiral acetate generation [ 5 ] .…”

Section: Assay Of Oxalacetase Activitymentioning

confidence: 99%

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Partial Purification and Some Properties of Oxalacetase from Aspergillus niger

1976

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1. Oxalacetase from Aspergillus niger was found to be an inducible enzyme, the induction being dependent not only on neutralisation of the acidic growth medium but also on the presence of carbonate. An explanation is proposed.2. Three methods were established for the quantitative determination of oxalacetase activity. These are based on the determination of the product acetate, on the absorbance of oxaloacetate and on coupling the hydrolysis of oxaloacetate to the oxidation of malate by NAD in the presence of malate dehydrogenase.3. Oxalacetase was purified about 50-fold from cell-free extracts of A . niger and used to determine some of its properties such as kinetic constants. 2S-[U-'4C,3-2H2]Malate in the presence of oxalacetase, NAD and malate dehydrogenase was partially converted to acetate and oxalate. The 3H/'4C ratio of the isolated acetate was nearly twice as high as that of the malate used initially. The result demonstrates that the keto form of oxaloacetate, not the enol, is the substrate of the enzyme. by incubation with fumarase in normal and tritiated water, respectively. The isolated mixture 1, in the presence of oxalacetase, NAD and malate dehydrogenase was incubated in tritiated water l' or formation of acetate and oxalate; the isolated mixture 2 was treated likewise in normal water.6. The mixtures of symmetrically labelled [3Ht]acetate and chiral acetates thus produced were isolated and the configuration of the [2H1 ,3Hl]acetate specimens was determined in the sequence acetate -+ malate + fumarate, as usual. The L2HI, 3Hl]acetate derived from 2S, 3S-[3-'Hl]malate (present in mixture 1) yielded a malate which on incubation with fumarase retained 65.0':;) of its total tritium content. This chiral acetate, therefore, had the R configuration. The [2H1,3Hl]acetate derived from 2S,3R-[2-2H1, 3-3Hl]malate produced a malate which retained 35 7" of its total tritium content, and therefore had the S configuration.7. It was concluded that the detachment of the oxaloyl residue from oxaloacetate and its replacement by a proton proceed with inversion of configuration at the methylene group which becomes methyl during the hydrolysis These results are taken from the Thesis of H. Lenz, Universitiit Regensburg, 1973; a preliminary account of part of this work has appeared elsewhere [I].

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“…Based on these results the enzyme more recently was applied to establish a configurational assay of chiral methyl groups [3-51. The method has since widely been used to elucidate the stereochemical course of enzymic reactions [6] including that of malate synthase itself [7]. Connected to these studies one of us (G. B) [8] had purified the synthase (about 300 mg) from baker's yeast (10 kg).…”

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confidence: 99%

Large‐Scale Purification and Some Properties of Malate Synthase from Baker's Yeast

Durchschlag¹,

Biedermann²,

Eggerer³

1981

European Journal of Biochemistry

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Malate synthase from baker's yeast (5 kg) was purified 400–500‐fold to homogeneity. About 50–200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell‐free crude extracts. The enzyme, pl = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies. Sedimentation and diffusion coefficients were determined: . The molecular weight of the synthase was found to be 175000 ± 10000 and 180000 ± 10000 by sedimentation/diffusion and by high‐speed sedimentation equilibrium respectively. It was concluded from these and other results that malate synthase has a molecular mass of 180000 ± 10000. The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66000. The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass. The binding of acetyl‐coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+. Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism. The intermolecular isotopic effect, kH : k2H= 1.4, was determined with acetyl‐coenzyme A and [2H3]acetylcoenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates. An enzymic procedure for the preparation of doubly labelled [3H,14C]acetyl‐coenzyme A is also presented.

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Enzymic Generation of Chiral Acetates. A Quantitative Evaluation of Their Configurational Assay

Cited by 33 publications

References 16 publications

Mechanistic and Stereochemical Studies on the Glycine Reductase of Clostridium sticklandii

Mechanistic and Stereochemical Studies on the Glycine Reductase of Clostridium sticklandii

Partial Purification and Some Properties of Oxalacetase from Aspergillus niger

Large‐Scale Purification and Some Properties of Malate Synthase from Baker's Yeast

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