Coenzyme active acetate

Chenoweth believes that an explanation of the above results may lie in the reactions occurring before the entrance of fatty acid metabolites into the citric acid cycle. Activated acetate, i.e. acetyl coenzyme A (AcCoA) is the end-product of fatty acid metabolism prior to its condensation with oxalacetate to form citrate. Possibly fluoro-fatty acids behave like non-fluorinated fatty acids. The end-product before the oxalacetate condensation could be the same for all three fluorinated inhibitors, viz. fluoroacetyl coenzyme A (FAcCoA). Fluorocitrate could then be formed by the condensation of oxalacetate with FAcCoA, thereby blocking the citric acid cycle. The specificity of antagonisms must therefore occur before entrance of the metabolites into the citric acid cycle. 

Soluble cytoplasmic sulfotrans-ferases conjugate activated sulfate (3 -phosphoadenine-5 -phosphosulfate) with alcohols and phenols. The conjugates are acids, as in the case of glucuronides. In this respect, they differ from conjugates formed by acetyltransfe-rases from activated acetate (acetyl-coenzyme A) and an alcohol or a phenol... 

Biotin enzymes are believed to function primarily in reversible carboxvlahon-decarboxylation reactions. For example, a biotin enzyme mediates the carboxylation of propionic acid to methylmalonic add, which is subsequently converted to succinic acid, a dtric acid cycle intermediate. A vitamin Bl2 coenzyme and coenzyme A are also essential to this overall reaction, again pointing out the interdependence of the B vitamin coenzymes. Another biotin enzyme-mediated reaction is the formation of malonyl-CoA by carboxylation of acetyl-CoA ( active acetate ). Malonyl-CoA is believed lo be a key intermediate in fatly add synthesis. 

TPP functions as a coenzyme which participates in decarboxylation of or-keto acids. Dehydrogenation and decarboxylation must precede the formation of active acetate in the initial reaction of the TCA cycle (citric acid cycle) ... 

The most important functions of pantothenic acid are its incorporation in coenzyme A and acyl carrier protein (AGP). Both CoA and AGP/4-phosphopantetheine function metabolically as carriers of acyl groups. Coenzyme A forms high-eneigy thioester bonds with carboxylic acids. The most important coenzyme is acetyl CoA. Acetic acid is produced during the metabolism of fatty acids, amino acids, or carbohydrates. The active acetate group of acetyl CoA can enter the Krebs cycle and is used in the synthesis of fatty acids or cholesterol. AGP is a component of the fatty acid synthase multienzyme complex. This complex catalyzes several reactions of fatty acid synthesis (condensation and reduction). The nature of the fatty acid synthase complex varies considerably among different species (91). 

The overall process leading to the formation of hydrogen bound to metabolic carriers can be separated into two main reaction sequences. Firstly, carbohydrates, amino acids and fatty acids, obtained by enzyme catalyzed cleavage of polysaccharides, proteins and lipids, respectively, are transformed into active acetic acid,CH3—CO—SCoA (where -SCoA represents coenzyme A). Secondly, the active acetic acid is degraded into C02 and metabolically bound hydrogen (symbolized by [H]) according to Eq.(32) ... 

It is well known that in the oxidation of pyruvate an active 2-carbon intermediate, known as active acetate, is formed (Gurin and Crandall, 1948 Bloch, 1948 Lehninger, 1950). A suggestion that pantothenic acid is involved in the conversion of acetate to citrate in yeast came from Novelli and Lipmann s (1950) experiments showing a correlation between the ability of the yeast to oxidize acetate or ethanol and the coenzyme A content of the cells. The formation of citrate from acetate and oxaloacetate in cell-free extracts from pigeon liver or yeast was also shown to require coenzyme A (Novelli and Lipmann, 1950 Stern and Ochoa, 1949, 1951). Further study of these enzyme systems led to the isolation of a crystalline condensing enzyme from pig heart (Ochoa et al., 1951) and to a formulation of the mechanism of the enzymatic synthesis of citric acid from... 

Since the original observation of Lipmann et cd. (1947) on the presence of pantothenic acid in coenzyme A, the purification and chemical structure of the coenzyme has been the subject of intensive investigation in many laboratories (Snell et al., 1950 Lynen and Reichert, 1951 Novell et al, 1951 Baddiley and Thain, 1951). As formulated at present, coenzyme A contains 1 adenine, 1 ribose, 1 sulfur, and 3 phosphates per pantothenate. Evidence presented by Lynen and Roichert (1951) indicates that acetyl-CoA, which is now considered synonomous with active acetate, is an acetylated mercaptan. A tentative structure of acetyl-CoA is given below. 

In 1951, Feodor Lynen (1911-1979) and his coworker E. Reichert demon-started that S-acetyl coenzyme A is a more generally implicated form of active acetate than acetyl phosphate that was recognized in this role by Fritz Lipmann in 1940. The thiol ester character of 5-acetyl Co A called the attention of Th. Wieland to energy-rich S-acyl compounds as promising intermediates for the formation of the peptide bond. In 1951, the same year when the isolation of 5-acetyl CoA was published [3], Wieland and his coworkers described [4] the preparation of thiophenyl esters of benzyloxycarbonyl-amino acids and benzyloxycarbonyl-peptides and their application in the synthesis of blocked peptides ... 

Many anaerobic microorganisms can use CO or CO2 as a sole source of carbon and CO and/or H2 for the generation of energy [1]. Thus, acetogens generate acetyl coenzyme A, an activated acetic acid that serves as a universal precursor for the generation of biomass (see below), and acetic acid from... 

Plants synthesize natural rubber via what is known as activated acetic acid, the acetic thioester of coenzyme A ... 

It was evident from early studies that in the presence of CoASH, ATP could in some way be used to activate acetate so that it will acetylate sulfanilamide (7) and choline (8). Chou and Lipmann (9) succeeded in partially purifying the enzyme(s) responsible for this activation from pigeon-liver extracts and they concluded that the phosphate bond energy of ATP is utilized to bring about the synthesis of acetyl coenzyme A (acetyl-SCoA) however, the mechanism of this activation remained obscure. The nature of the over-all process was further elucidated through the experiments of Lipmann et al. (10) who demonstrated that ATP, CoASH, and acetate react to form acetyl-SCoA, AMP, and inorganic pyrophosphate (P-P) in stoichiometric amounts [reaction (4)]. They also demonstrated that the reaction is freely reversible. More recently, the studies of Jones et al. (11) have indicated that the mechanism of this conversion is as follows ... 

Evidence for the formation of an activated succinate arising from a-keto-glutarate oxidation, which may be represented by a succinyl-coenzyme A complex, has also been presented by Sanadi and Littlefield working in Green s laboratory. These workers have found that sulfanilamide may be succinylated by a CoA-requiring mechanism, which appears to be essentially analogous to the acetylation of sulfanilamide by activated acetate. ... 

Barker has recently reviewed the metabolism of active acetate and has discussed the role of CoA in the various transformations of acetate. Only the more general developments regarding the mechanism of action of this coenzyme will be discussed. 

The 2-carbon unit or activated acetate, which is the acetylated form of coenzyme A, the pantothenic acid-containing enzyme, has been shown to occupy a central position in many converging pathways. The role of mitochondria and other essential cellular units in oxidative metabolism, which have been elucidated recently, link cytology and biochemistry closely together and may lead to closer correlation between pathologic anatomical changes demonstrable in tissue section and biochemical abnormalities found in body fluids and tissues (Chapter 12). 

The oxidation of pyruvic acid to form acetyl CoA was for many years the elusive active acetate . It was the target of active research in many laboratories since Peters demonstrated in the 1930s that cocarboxylase (thiamine pyrophosphate) is necessary for the process. The reaction implies an oxidative decarboxylation by which pyruvic acid loses CO2 and 2H to form acetyl CoA, and requires several enzymes and coenzymes (thiamine pyrophosphate, coenzyme A, NAD, lipoic acid and Mg +). 

Coenzyme A can form high-energy bonds with acetic acid via its sulfhydryl group to yield acetyl-coenzyme A (acetyl-CoA), also referred to as activated acetate. Through a variety of biochemical sequences, CoA can also be converted to a number of other acyl-CoA derivatives, such as malonyl-CoA, methylmalo-... 

Acetyl-CoA is the real form of activated acetate where 36.9 kJ/mol (8.8 kcal/mol) is liberated upon hydrolysis (see Chapter 2). Reduced lipoic add is reoxidized with an FAD coenzyme present on the complex. 

ACh synthesis is carried out in the neuron by means of the coenzyme A (CoA) and a specific enzyme, choline-acetylase (ChAc, choline-acetyltransferase), which transfers the acetyl radical from active acetate (acetyl CoA) to choline (see literature in ref. 16) ... 

Studies of the enzymic mechanism of the citric acid synthesis by Stern and Ochoa have directly shown that citric acid, and not aconitic acid, is the primary product. It had earlier been thought that the mechanism of citric acid synthesis might be similar to that of the reaction leading in vitro to the formation of citric acid from oxalacetic and pyruvic acid in the presence of hydrogen peroxide, where oxalocitramalic acid is an intermediate. Martins, however, found this substance to be metabolically inert in animal tissue. Stern and Ochoa found that aqueous extracts of acetone-dried pigeon liver formed citrate when acetate, oxalacetate, ATP, coenzyme A, and Mg or Mn ions were present. Thus the condensation reaction is preceded by the decarboxylation of pyruvic acid and the formation of an active form of acetate. This active acetate, as discussed below, is acetyl coenzyme A. 

The problem of the identification of the active acetic acid has recently been brought to its solution as a result of studies on the chemical nature of coenzyme A, which Lipmann discovered in 1945. Progress in this field has been due mainly to Lipmann, Snell, Stadtman, Ochoa, and Lynen. 

Stadtman, Novelli, and Lipmann and Korkes, Stern, Gunsalus, and Ochoa concluded that active acetate is acetyl coenzyme A. 

A major advance was the isolation from yeast cells by Lynen and Reichert in 1951 of an acetylated coenzyme A derivative and the demonstration that the acetyl group is in all probability attached to the sulfur atom. As already mentioned, the existence of an acetylated coenzyme A had already been postulated before,but the acetylated form had not been isolated, nor was there any indication regarding the type of acetyl compound involved. Lynen succeeded in isolating active acetic acid from yeast cells which were allowed to respire in ethanol and acetic acid, killed by boiling, and extracted with phenol. The phenol extract after addition of ether was extracted with water, and the concentrated aqueous solution was treated with barium. The further fractionation of the barium salts employing adsorption on active charcoal, elution and precipitation by acetone, yielded a compound which showed the following properties ... 

Further convincing proof for the reality of active acetic acid has been supplied by Stern, Shapiro, Stadtman, and Ochoa. These authors tested a sample of acetyl coenzyme A which Lynen had assayed by the sulfanilamide test. When this product was added to oxalacetate in the... 

There is thus powerful evidence in support of the view that the thiol group is the prosthetic group of coenzyme A, and that active acetic acid is the S-acetylated coenzyme A. 

Isotope experiments of Weinhouse, Medes, and Floyd and of Buchanan, Sakami, and Gurin, have shown that acetoacetic acid and other /3-ketonic acids can supply active acetic acid for the synthesis of citric acid. Since the synthesis of citrate requires acetyl coenzyme A, the breakdown of 8-ketonic acids must lead to the formation of acetyl coenzyme A. Lynen and Reichert assumed a thioclastic fission, as formulated in Scheme 8. Observations which support this... 

Fig. 56. Coenzymes which take part in the formation of active acetate (cf. Fig. 57 for the function of flavoprotein and NAD" ).

HS-CoA Coenzyme A. HS-CoA accepts the acetyl residue with its sulphydryl (HS) group. The acetyl residue linked to CoA through an energy-rich thiol ester bond is known as acetyl CoA or active acetate. This active acetate is a key substance in metabolism, to which we shall often have need to return. We are indebted to Lynen for a major part of our knowledge of this nodal point of metabolism. 

We must now discuss the regeneration of LAA. The liberation of reduced LAA, which bears two HS-groups, occurs simultaneously with the formation of active acetate. Removal of 2H leads to the reformation of the oxidized form of LAA with an intact disulphide ring. Initially the hydrogen atoms are accepted by a flavoprotein, which transfers them to NAD+. The NADH -F H+so formed can then enter the respiratory chain where it is utilized for ATP formation. We have discussed the decarboxylation of pyruvate in some detail. As justification for this it should be pointed out that thiamine is identical with vitamin Bj. Thus, thiamine pyrophosphate serves as a good example of the kinds of function vitamins can exercise they can be coenzymes or constituents of coenzymes. In addition, we shall note other cases of decarboxylation which proceed, in part, according to the same mechanism. Two instances are alcoholic fermentation and the decarboxylation of a-ketoglutarate in the citric acid cycle which we come to now. 

---