Coenzyme isolation

Nicotinic acid (niacin) was prepared first by oxidation of the alkaloid nicotine, but nut until 1913 was it isolated from yeast and recognized as an essential food factor (see tlu structures). In 1934-1935, nicotinamide was obtained from the hydrolysis of a coenzyme isolated from horse red blood cells. This coenzyme was later named coenzyme II and is now more commonly called nicolinainide adenine dinuclen-tide pho.iphate (NADP). 

The coenzyme isolated from yeast - has been assigned the structure of a glucose-l-phosphate molecule attached to uridine-5 -phosphate through a pyrophosphate link (Fig. 7). It has been abbreviated as UDPG. The discovery of a uridine-5 -phosphate in this compound preceded the recent demonstration of such a moiety in ribose nucleic acid. 

Discovery of the connection between vitamins and coenzymes. Isolation of the pyridine and flavin coenzymes (v. Euler, Theorell, Warburg)... 

Coenzyme A was isolated and identified by Fritz Lipmann an American bio chemist Lipmann shared the 1953 Nobel Prize in physiol ogy or medicine for this work... 

Cysteine [52-90 ] is a thiol-bearing amino acid which is readily isolated from the hydrolysis of protein. There ate only small amounts of cysteine and its disulfide, cystine, in living tissue (7). Glutathione [70-18-8] contains a mercaptomethyl group, HSCH2, and is a commonly found tripeptide in plants and animals. Coenzyme A [85-61-0] is another naturally occurring thiol that plays a central role in the synthesis and degradation of fatty acids. 

Anhydrotetracycline oxygenase from Streptomjces aureofaciens which cataly2es the conversion of anhydrotetracycline to dehydrotetracycline, has been isolated and characterized as a flavin-dependent oxygenase (83). It consists of two subunits of mol wt = 57, 500 based on SDS/polyacrylamide—gel electrophoresis. The cosynthetic factor 1 of Streptomjces aureofaciens involved in the reduction of 5a,lla-dehydrochlortetracycline to chlortetracycline, has been identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin. This work was aided by comparison of spectral data with that of an authentic sample obtained from the hydrolysis of coenzyme F-420 (84). 

Riboflavin was first isolated from whey in 1879 by Blyth, and the structure was determined by Kuhn and coworkers in 1933. For the structure determination, this group isolated 30 mg of pure riboflavin from the whites of about 10,000 eggs. The discovery of the actions of riboflavin in biological systems arose from the work of Otto Warburg in Germany and Hugo Theorell in Sweden, both of whom identified yellow substances bound to a yeast enzyme involved in the oxidation of pyridine nucleotides. Theorell showed that riboflavin 5 -phosphate was the source of the yellow color in this old yellow enzyme. By 1938, Warburg had identified FAD, the second common form of riboflavin, as the coenzyme in D-amino acid oxidase, another yellow protein. Riboflavin deficiencies are not at all common. Humans require only about 2 mg per day, and the vitamin is prevalent in many foods. This vitamin... 

Divalent sulfur compounds are achiral, but trivalent sulfur compounds called sulfonium stilts (R3S+) can be chiral. Like phosphines, sulfonium salts undergo relatively slow inversion, so chiral sulfonium salts are configurationally stable and can be isolated. The best known example is the coenzyme 5-adenosylmethionine, the so-called biological methyl donor, which is involved in many metabolic pathways as a source of CH3 groups. (The S" in the name S-adenosylmethionine stands for sulfur and means that the adeno-syl group is attached to the sulfur atom of methionine.) The molecule has S stereochemistry at sulfur ana is configurationally stable for several days at room temperature. Jts R enantiomer is also known but has no biological activity. 

Figure 8.27 Reduction of aldehyde in SCCO2 by an isolated enzyme, horse liver alcohol dehydrogenase (HLADH) [20c] (a) Reaction scheme (b) fluorinated coenzyme soluble in CO2 and (c) effect of coenzyme on the reaction.

A new, heat-stable, coenzyme concerned with methyl group transfer has been isolated from Methanobacterium. The coenzyme, which is involved in transmethylation reactions prior to methane formation by the organism, contains phosphorus and has a u.v. absorption at 260 nm, suggesting that it may be a nucleotide. 

Song B, BB Ward (2005) Genetic diversity of benzoyl coenzyme A reductase genes detected in denitrifying isolates and estuarine sediment communities. A/ / / Environ Microbiol 71 2036-2045. 

Naturally, the biosynthesis of cobalamins themselves require delivery of Co ions at a particular point in the reaction scheme. Cobaltochelatase catalyzes the ATP-dependent insertion of Co11 into the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrifleans. Cobaltochelatase is a heterodimeric enzyme (140 KDA and 450 KDA subunits each inactive in isolation), and the two components have been isolated and purified to homogeneity.1119 The reaction product is divalent cobyrinic acid, demonstrating that hydrogenobyrinic acid and its diamide (255) are precursors of AdoCbl. 

In 1958 Barker (20) isolated a red, heat stable, light labile, cofactor which was required for the metabolism of glutamate in cell-extracts of Clostridium tetanomorphum. Subsequently this cofactor was crystallized. X-ray crystallography identified Barker s cofactor as the coenzyme form of Vitamin B12 (15, 21). 

The very high resolution for the ESR spectrum of cob(II)alamin in the enzyme system is undoubtedly due to the fact that all the coenzyme molecules are bound in an identical environment at the enzyme active site. This results in a homogeneous cobalt-benzimidazole geometry, because both identical binding sites, solvent, and solute molecules can no longer approach the Bia-molecule closely. In addition, the enzyme bound cob(II)alamin molecules are more isolated from one another and thus relaxation due to spin-spin interactions is less effective in broadening spectral lines. 

Page 243, line 6. L-Guluronic acid has been isolated from alginic acid.264(d Page 244, line 5 up. Read coenzymes.259W... 

Brain ChAT has a KD for choline of approximately 1 mmol/1 and for acetyl coenzyme A (CoA) of approximately 10pmol/l. The activity of the isolated enzyme, assayed in the presence of optimal concentrations of cofactors and substrates, appears far greater than the rate at which choline is converted to ACh in vivo. This suggests that the activity of ChAT is repressed in vivo. Surprisingly, inhibitors of ChAT do not decrease ACh synthesis when used in vivo this may reflect a failure to achieve a sufficient local concentration of inhibitor, but also suggests that this step is not rate-limiting in the synthesis of ACh [18-20]. 

Abnormalities of the respiratoiy chain. These are increasingly identified as the hallmark of mitochondrial diseases or mitochondrial encephalomyopathies [13]. They can be identified on the basis of polarographic studies showing differential impairment in the ability of isolated intact mitochondria to use different substrates. For example, defective respiration with NAD-dependent substrates, such as pyruvate and malate, but normal respiration with FAD-dependent substrates, such as succinate, suggests an isolated defect of complex I (Fig. 42-3). However, defective respiration with both types of substrates in the presence of normal cytochrome c oxidase activity, also termed complex IV, localizes the lesions to complex III (Fig. 42-3). Because frozen muscle is much more commonly available than fresh tissue, electron transport is usually measured through discrete portions of the respiratory chain. Thus, isolated defects of NADH-cytochrome c reductase, or NADH-coenzyme Q (CoQ) reductase suggest a problem within complex I, while a simultaneous defect of NADH and succinate-cytochrome c reductase activities points to a biochemical error in complex III (Fig. 42-3). Isolated defects of complex III can be confirmed by measuring reduced CoQ-cytochrome c reductase activity. 

The leucocyte method estimates pyridoxal phosphate in isolated leucocytes it is based on a coenzyme-catalyzed tyrosine decarboxylase system from S. faecalis (B32). Enough data are not yet on hand to evaluate this method. The determination of circulating or available vitamin Ba should offer a more direct approach. 

Knowledge of the coenzyme forms of vitamin Bi2 has increased steadily. The first coenzyme of Bi2 isolated from bacteria had similarities to pseudovitamin Bi2 it contained adenylic acid instead of 5,6-dimethyl-benzimidazole, but differed in lacking cyanide and having an extra molecule of adenine which was assumed to be bound to the cobalt atom by the coordination site, often occupied by cyanide (B24). This coenzyme, adenylcobamide, was completely inactive for Ochromonas malhamensis, but active for Escherichia coli 113-3. 

Ochromonas malhamensis responds to vitamin Bi2, but not to pseudovitamin Bi2 E. coli responds to both forms. Later other coenzymes were isolated (B25). One contained benzimidazole (BC), the other... 

The first of these new, electron transferring components was coenzyme Q (CoQ). Festenstein in R.A. Morton s laboratory in Liverpool had isolated crude preparations from intestinal mucosa in 1955. Purer material was obtained the next year from rat liver by Morton. The material was lipid soluble, widely distributed, and had the properties of a quinone and so was initially called ubiquinone. Its function was unclear. At the same time Crane, Hatefi and Lester in Wisconsin were trying to identify the substances in the electron transport chain acting between NADH and cytochrome b. Using lipid extractants they isolated a new quininoid coenzyme which showed redox changes in respiration. They called it coenzyme Q (CoQ). CoQ was later shown to be identical to ubiquinone. 

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