Studies on Cyclic Anhydrides. II. Equilibrium Constants for the Equilibrium between Acid and Anhydride in Aqueous Solution of Some Alkylsubstituted Maleic Acids.

A continuous spectrophotonietric assay was developed for the adenosylcobalamin -dependent 2-methyleneglutarate mutase from Clostridium harkeri. Thereby the product (R)-3-methylitaconate is converted by the A-isomerase Ii-om the same organism to 2,3-dimethylmaleate which absorbs at 240 nm, much higher than both parent compounds (Ac = 3.7 mM-' cm-'). In addition a discontinuous assay using the facile formation of 2,3-dimethylmaleic anhydride in aqueous solution at pH 0-1 (Ac = 4.0 mM-' . cm-' at 256 nm) was established. The mutase and the isomerase were purified together by chromatography on quaternary-amineSepharose (Q-Sepharose) and on cyanocobalamin-agarose. The enzymes were separated and obtained in homogenous forms by preparative PAGE in non-denaturing buffer. Both enzymes appear to be homotetramers with subunits of 70 kDa (mutase) and 50 kDa (isomerase). The equilibrium constants for both reactions were determined at 1 = 0.1 M and 25°C:The strict anaerobic eubacterium Clostridium harkeri ferments nicotinic acid to ammonia, propionate, acetate and carbon dioxide [l]. A key reaction of this pathway is the reversible rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate which is dependent on adenosylcobalaniin [2-41, (Scheme 1).During this reaction, which is catalyzed by 2-methyleneglutarate mutase, the acrylate residue is shifted from the carbon to the M carbon of a propionate residue while a hydrogen moves in the opposite direction.In a first attempt to elucidate the mechanism of this remarkable interconversion, the steric course at the CI carbons of the substrates was determined. Therefore (R)-3-methyl-[3-2H]itaconate was converted enzymatically to (S)-2-methylene [4-'H ,]glutarate demonstrating inversion of configuration at the M carbon [5]. This result is of considerable interest since the closely related glutainate mutase also works with inversion [6, 71 whereas with methylmalonyl-CoA mutase retention was found [8]. Recent progress with chemical model reactions show that this type of reactions proceeds via free radicals [9 -121 probably with correspondingly substituted cyclopropylmethylene radicals as intermediates [lo, 111. In order to test this hypothesis purified enzymes are required. However, 2-methyleneglutarate mutase is not available in pure form [3]. The experiments described in this paper fill this gap. A new assay for this enzyme, its purification to homogeneity, and the determination of the equilibrium constant of the mutase reaction will be presented. In the course of this study 3-methylitaconate A-isomerase, catalyzing the consecutive step in nicotinate fermentation yielding dimethylmaleate, was also purified (Scheme 2). MATERIALS AND METHODSSources and synthesis of 2-methyleneglutarate, (R,S)-3-methylitaconate and 2,3-dimethylmaleic anhydride as well as growth of C. harkeri (DSM 1233) were described previously [5]. Aqueous solutions of 2,3-dimethylmaleate were prepared by dissolving the anhydride in an excess of NaOH. Then the solution was adjusted to the desired pH. (R,S)-3-Methyl...

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“…[acid]-' = S [21]. This high equilibrium constant is remarkable when it is compared with that of succinic acid, K = 8 x [22].…”

Section: Assay Of2-rnethyleneglutarate Rnutase and 3-rnethylitaconatementioning

confidence: 90%

Assay and purification of the adenosylcobalamin‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri

Michel

Hartrampf

1989

European Journal of Biochemistry

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“…The expected change in absorption is seen most clearly (208 to 218 nm) in the case of the cyclohexene-l,2-dioic system (42a). The positions of the water-mediated equilibria between the enedioic acids and the corresponding anhydrides have been determined by Eberson (1964) by a pH-stat method for mono and dialkylmaleic and cyclohex-l,2-dioic anhydrides and, for the last mentioned compound, also by measurement of the intensity of absorption at 256 nm, Craig's observation with 42b and ours with 42a accord with Eberson's finding that the diacid is the highly favoured species in the equilibrium of cyclehexane-Lz-dioic anhydride in that the absorption due to the acid-ester obscures that due to the ene-anhydride in the aged alcoholic solutions in both instances.…”

Section: MMmentioning

confidence: 99%

Steric effects on the ultraviolet absorption of 1,2-enedioic and related systems

Balasubrahmanyam

Rao

1978

Proc. Indian Acad. Sci., A (Chemical Sciences)

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l,2-Enedioic systems, being sterically perturbed from planarity do not show the effect of the extended conjugation expected of a (formal) trienic entity. In the absence of a model which approximates to a uniplanar situation, the strategy of replacing an ester group in the enedioates by a cyano (for which less stringent steric demand may be presumed) and noting the correction concomitant to this replacement was adopted to arrive at a notional figure for the position of maximal absorption in the planar enedioates. From this the conclusion, subject to substantiation by molecular mechanical or quantum chemical calculations, was drawn that even the E-isomeric and comparatively less substituted enedioates are highly sterically perturbed. An alternative to an earlier explanation of the bathochromic shift of absorption maxima encountered in the 5-cyclicene-ester and ene-nitrile, relative to the 6-cyclic analogues (observed also with the enedioates and cyanovinyl ester systems), seen later to have been based on unwarranted premises, has been advanced. A comment on the absorption characteristics of enedioic anhydrides has been appended.

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“…(1)], have been determined in several cases. [9][10][11][12] The values of K E vary from 10 Ϫ7 to 25 depending on the structure of the diacid. The unionized acid is the kinetically active species in anhydride formation.…”

Section: Introductionmentioning

confidence: 99%

Hydrolysis of 1,8- and 2,3-naphthalic anhydrides and the mechanism of cyclization of 1,8-naphthalic acid in aqueous solutionsThe IUPAC name for naphthalic acid is napthalenedicarboxylic acid.Electronic supplementary information (ESI) available: tables containing the values of the rate constants. See http://www.rsc.org/suppdata/p2/b1/b104148g/

Barros

Yunes

Menegon

et al. 2001

J. Chem. Soc., Perkin Trans. 2

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Studies on Cyclic Anhydrides. II. Equilibrium Constants for the Equilibrium between Acid and Anhydride in Aqueous Solution of Some Alkylsubstituted Maleic Acids.

Cited by 16 publications

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Assay and purification of the adenosylcobalamin‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri

Assay and purification of the adenosylcobalamin‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri

Steric effects on the ultraviolet absorption of 1,2-enedioic and related systems

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