4-Hydroxy -aconitate

Water can add reversibly to o ,/3-unsalurated aldehydes and ketones to yield /3-hydroxy aldehydes and ketones, although the position of the equilibrium generally favors unsaturated reactant rather than saturated adduct. A related addition to an c /S-unsaturated carboxylic acid occurs in numerous biological pathways, such as the citric acid cycle of food metabolism where ds-aconitate is converted into isocitrate by conjugate addition of water to a double bond. 

Step 2 of Figure 29.12 Isomerization Citrate, a prochiral tertiary alcohol, is next converted into its isomer, (2, 35)-isocitrate, a chiral secondary alcohol. The isomerization occurs in two steps, both of which are catalyzed by the same aconitase enzyme. The initial step is an ElcB dehydration of a /3-hydroxy acid to give cfs-aconitate, the same sort of reaction that occurs in step 9 of glycolysis (Figure 29.7). The second step is a conjugate nucleophilic addition of water to the C=C bond (Section 19.13). The dehydration of citrate takes place specifically on the pro-R arm—the one derived from oxaloacetate—rather than on the pro-S arm derived from acetyl CoA. 

Acetic, aconitic, ascorbic, benzoic, butyric, caffeic, citric, /j-coumaric, ferulic, fumaric, glutaric, glycolic, glyoxilic, malic, malonic, oxalacetic, oxalic, p-hydroxy benzoic, propionic, succinic, syringic, tartaric, valeric, vanillic Fatty acids... 

Significant inhibition of all activities below pH 7 by citrate for which a Ki value of 5.2 mil/ has been observed at pH 6.5. Isocitrate much less effective, and only minor effects noted with cis-aconitate, f-malate, succinate, ar ketoglutarate, and oxalate. Malonate, tartrate, L-glutamate, glutarate, adipate, 0-hydroxy-0-methyl glutaryl-CoA, and 0-hydroxybutyrate also found ineffective as instantaneous inhibitors... 

Bromate, chloride, bromide, nitrite, nitrate, hypophosphite (HP022 ), selenite, selenate, sulphate, phosphate, pyrophosphate, arsenate, chromate, a-hydroxybutyrate, butyrate, formate, acetate, glycolate, gluconate, valerate, a-hydroxy valerate, pyruvate, monochloroacetate, dichloroacetate, trifluoroacetate, galactonurate, gluconurate, a-keto-glutarate, oxalate, fumarate, phthalate, oxalacetate, citrate, isocitrate, cis aconitate, trans aconitate, succinate, maleate, malonate, quinate, tartrate, hexane sulphonate, octane sulphonate, octane sulphate, decane sulphonate, dodecane sulphonate and dodecane sulphate... 

IV. TRI-BASIC ACIDS AND HYDROXY TRI-BASIC ACIDS Tri-carballylic Acid and Aconitic Acid... 

Citrate is not a good substrate for decarboxylation. Decarboxylation is usually carried out on alpha-keto acids (like pyruvate, above) or alpha-hydroxy acids. Conversion of citrate into an alpha-hydroxy acid involves a two-step process of water removal (dehydration), making a double bond, and readdition (hydration) of the intermediate—aconitate as Figure 10-4 shows. The enzyme responsible for this isomerization is aconitase. 

Acetal Acetamide Acetone sodium bisulfite Acetophenone Aconitic acid Acrylonitrile Adipamide Alcohol Alloxan monohydrate Allyl amine Allyl anthranilate 2-Aminobenzene sulfonic acid 4-Amino-4 -hydroxy-3-methyldiphenylamine Aminomethyl propanediol Aminomethyl propanol Ammonium hydroxide Arachidic acid Arsine Azelaic acid Benzil... 

Fig. 7.2 Chromatogram of acidic metabolites extracted from the urine of a normal child using DEAE-Sephadex and re-extraction with solvents after ethoxime formation and freeze-drying by reconstitution in water, acidification with hydrochloric acid, saturation with sodium chloride, and solvent extraction with diethyl ether (three times) and ethyl acetate (three times), evaporation of the solvents from the combined extracts using dry nitrogen and trimethylsilylation using the minimum quantity of BSTFA. Separated on 10 per cent OV-101 on HP Chromosorb W (80-100 mesh) by temperature programming from 110°C to 285°C at 4°C min with an initial 5 min isothermal delay. Peak identifications are 1, phenol plus lactate 2, glycollate 3, cresol 4, 3-hydroxyisovalerate 5, benzoate 6, phosphate 7, succinate 8, 3-methyladipate 9, 3-hydroxy-3-methyl-glutarate 10, 4-hydroxyphenylacetate 11, homovanillate plus some aconitate 12, hippurate 13, citrate 14, vanilmandelate 15, n-tetracosane (standard) 16, n-hexacosane (standard).

Fig. 16.13 Chromatograms of organic acids extracted using DEAE-Sephadex (upper) and DEAE-Sephadex with solvent re-extraction (lower) from an infant with hereditary tyrosinaemia type I. Separated as their ethoxime and trimethylsilyl derivatives on 10 per cent OV-101 on HP Chromosorb W (80-100 mesh) using temperature programming from 110°C to 285°C at 4°C min with a 5 min initial isothermal delay. Peak identifications are 1, lactate 2, pyruvate 3, sulphate 4, phosphate 5, succinate 6, tetronates 7, aconitate plus homovanillate 8, hippurate 9, citrate plus isocitrate 10, 4-hydroxy-phenyl-lactate 11, 4-hydroxyphenylpyruvate (ethoxime) 12, vanil-lactate 13, 4-hydroxyphenylpyruvate (tri-TMS) 14, urate 15, n-tetracosane (standard) 16, n-hexacosane (standard). (The horizontal axes represent the time elapsed in minutes from sample injection.) The predominance of 4-hydroxyphenyl-lactate and the absence of 4-hydroxyphenylacetate is notable. There is no evidence for the metabolites observed by Lindblad etfl/. (1977) (see Fig. 16.14).

Lactate, glycolate, succinate, chloride, malate, sulphate, tartrate, maleate, fluoride, a-hydroxybutyrate, hydroxy valerate, formate, valerate, pyruvate, monochloroacetate, bromate, galaconurate, nitrite, gluconurate, dichloroacetate, trifuoroacetate, hypophosphite, selenite, bromide, nitrate, oxalate, selenate, a-ketoglutarate, fumarate, phthalate, oxalacetate, phosphate, arsenate, chromate, citrate, isocitrate, cis aconitate and transaconitrate... 

---