Acetate, active from citrate

In 1932 Krebs was studying the rates of oxidation of small organic acids by kidney and liver tissue. Only a few substances were active in these experiments —notably succinate, fumarate, acetate, malate, and citrate (Figure 20.2). Later it was found that oxaloacetate could be made from pyruvate in such tissues, and that it could be further oxidized like the other dicarboxylic acids. 

Albersheim and Killias 167) purified the enzyme ninefold from Pectinol R-10. The optimal pH for activity was 5.1-5.2, and its isoelectric point was between 3 and 4. Pectin lyase was active on 65% esterified citrus pectin but not on polygalacturonic acid. The enzyme was more active in citrate and phosphate buflFers than in acetate. The addition of CaCL> to reaction mixtures buflFered with citrate or phosphate inhibited the reaction. Product inhibition was markedly increased by addition of plant auxins 168). Bull 169) suggested that the auxin eflFects were artifacts in spectrophometrically dense reaction mixtures. 

Comparatively, less-studied system is the peroxidase-catalyzed oxidations. One such example is the enantioselective oxidation of phenylmethylsulfide catalyzed by chloroperoxidase from Caldariomyces fumago in several buffer-IL mixtures at different pH values (Scheme 10.12) [107]. In this case, the chloroperoxidase-catalyzed sulfoxidation showed 70% product yield and above 99% ee, in ILs like [MMIM] [Me2P04] and cholinium acetate and cholinium citrate. But for the same sulfoxidation reaction catalyzed by chloroperoxidase, complete loss of enzyme activity was observed in morpholine containing ILs and [MMIM][MeSOJ [107]. The authors have pointed out that the addition of IL to the reaction medium influences the pH level and enzyme activity. For example, addition of 30% (vol/vol) [MMIM] [Me PO J increases the pH of a potassium phosphate buffer solution from 2.7 to 3.7. Enzyme activity of chloroperoxidase was significantly reduced at pH 2.7 which was recovered by increasing the pH to 3.7. (Note The authors have chosen potassium... 

Energy Balance of the Aerobic Carbohydrate Breakdown. Up to the formation of pyruvate 1 mole of ATP and 1 of NADHa arise from each triose. Another mole of NAD is reduced during the oxidative decarboxylation of pyruvate to acetyl-CoA. Up to the formation of acetyl-CoA (employing the respiratory chain) 1 - - 2 X 3 = 7 ATP are stored. Complete oxidation of active acetate in the citrate cycle yields another 12 moles of ATP per triose, i.e. a total of 19 ATP per mole of triose or 38 per mole of glucose. 

The citrate cycle is indeed the pivot on which the metabolic processes revolve, with respect both to the terminal oxidation of foodstuffs and to the synthetic activities. The citrate cycle requires acetyl-CoA, which is also the universal starting material for the synthesis of endogenous substances. Acetyl-CoA (active acetate) is formed mainly in two reaction sequences from the breakdown of fatty acids and from the breakdown of carbohydrates. The various interrelationships have been represented graphically on the fold-out chart in the back of this book. 

Among the most deadly of simple compounds is sodium fluoroacetate. The LD50 (the dose lethal for 50% of animals receiving it) is only 0.2 mg/kg for rats, over tenfold less than that of the nerve poison diisopropylphosphofluoridate (Chapter 12).a b Popular, but controversial, as the rodent poison "1080," fluoroacetate is also found in the leaves of several poisonous plants in Africa, Australia, and South America. Surprisingly, difluoroacetate HCF2-COO is nontoxic and biochemical studies reveal that monofluoroacetate has no toxic effect on cells until it is converted metabolically in a "lethal synthesis" to 2R,3R-2-fluorocitrate, which is a competitive inhibitor of aconitase (aconitate hydratase, Eq. 13-17).b This fact was difficult to understand since citrate formed by the reaction of fluorooxalo-acetate and acetyl-CoA has only weak inhibitory activity toward the same enzyme. Yet, it is the fluorocitrate formed from fluorooxaloacetate that contains a fluorine atom at a site that is attacked by aconitase in the citric acid cycle. 

Fig. 1. Prostatic acid phosphatase activity as a function of pH ( ) phenyl phosphate (O) p-nitrophenyl phosphate and (A) /8-glycerophosphate. Buffers Ac, acetate Cit, citrate and tris. From Nigam et al. (88).

The dialyzed enzyme solution was now subjected to a repetition of the preceding procedures admixture of suflBcient calcium phosphate gel to adsorb protein but leave the enzyme in solution centrifugation and addition of more gel to the supernatant to adsorb the enzyme elution of the enzyme from the gel with a mixture of 0.15 M acetate and 0.015 M citrate at pH 4.5 addition of solid ammonium sulfate to the eluate to 55% saturation and precipitation of the enzyme. At this stage, the purifications ranged from 650- to 1100-fold with a recovery of approximately 20-30% of the activity present in the crude red cell hemolysate. Solution of this precipitate, dialysis treatment with solid ammonium sulfate and collection of the precipitate appearing between 40 and 55% saturation yielded a preparation that represented a 1500-fold purification. The preparations were stable when left sedimented in the ammonium sulfate solution. 

The biosynthetic reactions involve a series of condensation processes and are distributed between cytosol and microsomes. All of the carbons of cholesterol are derived from acetyl-CoA, 15 from the methyl and 12 from the carboxyl carbon atoms. Acetyl-CoA is derived from mitochondrial oxidation of metabolic fuels (e.g., fatty acids) and transported to cytosol as citrate (Chapter 18) or by activation of acetate (e.g., derived from ethanol oxidation) by cytosolic acetyl-CoA synthase (Chapter 18). All of the reducing equivalents are provided by NADPH. 

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