Thioester-biotin

The mechanism of the C02 transfer reaction with acetyl CoA to give mal-onyl CoA is thought to involve C02 as the reactive species. One proposal is that loss of C02 is favored by hydrogen-bond formation between the A -carboxy-biotin carbonyl group and a nearby acidic site in the enzyme. Simultaneous deprotonation of acetyl CoA by a basic site in the enzyme gives a thioester eno-late ion that can react with C02 as it is formed (Figure 29.6). 

In the degradation of isoleucine, (3 oxidation proceeds to completion in the normal way with generation of acetyl-CoA and propionyl-CoA. However, in the catabolism of leucine after the initial dehydrogenation in the (3-oxidation sequence, carbon dioxide is added using a biotin enzyme (Chapter 14). The double bond conjugated with the carbonyl of the thioester makes this carboxylation analogous to a standard (3-carboxylation reaction. Why add the extra C02 ... 

Thiol groups are introduced into antigens by reaction of their primary amino groups with SATA. This is the same kind of reaction as that used for incorporation of biotin molecules into antigens or of SMCC into AChE and requires the same precautions. The thioester function of SATA is then hydrolyzed in the presence of hydroxylamme in order to reveal the thiol groups. Excess reagents (SATA, hydroxylamme) are finally eliminated by molecular sieve chro-... 

Biotin is the coenzyme in a small number of carboxylation reactions in mammalian metabolism and some decarboxylation and transcarboxylation reactions in bacteria. Although the biotin-dependent enzymes are cytosolic and mitochondrial, about 25% of tissue biotin is found in the nucleus, much of it bound as thioesters to histones. Biotin has two noncoenzyme functions induction of enzyme synthesis and regulation of the cell cycle. 

Biotinidase is also the major plasma binding protein for biotin. The pH optimum of the enzyme is 4.5 to 5.5, and its is in the micromolar range, compared with the nanomolar concentrations of biocytin, so it will have little enzymic activity in plasma. Rather, it functions as a transport protein for biotin, preventing its urinary excretion children with biotinidase deficiency (Section 11.2.3.1) excrete large amounts of both biocytin and free biotin. Biotin is covalently bound to biotinidase in plasma, as a thioester to a cysteine residue in the active site of the enzyme (see Figure 11.1). This thioester is formed only from biocytin, not free biotin, and is presumably the (normally transient)... 

In addition to phenylacetonitrile and fluorene, various active methylene compounds such as indene, propiophenone, phenyl propionate, benzyl phenylacetate afforded the corresponding carboxylated products by the carboxylation reaction with La(0 Pr)3-Ph-N=C=0-C02 system. Of fundamental and practical importance is that S-benzyl thiopropionate was effectively carboxylated into a thioester of 2-methylmalonate in a good yield, since this reaction is related to the biological carboxylation of propionyl coenzyme A with a biotin enzyme. Other thioesters were also carboxylated similarly, where successful examples were thioesters of phenylacetic, acetic, and isovaleric acids carrying active methylene and methyne groups, respectively. 

The a-oxoamine synthases family is a small group of fold-type I enzymes that catalyze Claisen condensations between amino acids and acyl-CoA thioesters (Figure 16). Members of this family are (1) 8-amino-7-oxononanoate (AON) synthase (AONS), which catalyzes the first committed step in the biosynthesis of biotine, (2) 5-aminolevulinate synthase (ALAS), responsible for the condensation between glycine and succinyl-CoA, which yields aminolevulinate, the universal precursor of tetrapyrrolic compounds, (3) serine palmitoyltransferase (SPT), which catalyzes the first reaction in sphingolipids synthesis, and (4) 2-amino-3-ketobutyrate CoA ligase (KBL), involved in the threonine degradation pathway. With the exception of the reaction catalyzed by KLB, all condensation reactions involve a decarboxylase step. 

Apart from the true Claisen condensations discussed in the previous section in which the electrophilic reaction partner is another thioester, a number of enzymes also catalyze related Claisen-like condensations in which an acyl-CoA-based nucleophile reacts with other electrophilic carbonyl groups such as ketones, aldehydes, and the carboxylate group of carboxy-biotin. The most important examples of such enzymes are hydroxymethylglutaryl-CoA (HMG-CoA) synthase, citrate and homocitrate synthase (HCS), malate and ct-isopropylmalate synthase (ct-IPMS), and the biotin-dependent acetyl- and propionyl-CoA carboxylases. 

Fig. 8.12. CoA and biotin, activation-transfer coenzymes. A. Coenzyme A (CoA or CoASH) and phosphopantetheine are synthesized from the vitamin pantothenate (pantothenic acid). The active sulfhydryl group, shown in blue, binds to acyl groups (e.g., acetyl, succinyl, or fatty acyl) to form thioesters. B. Biotin activates and transfers CO2 to compounds in car-boxylation reactions. The reactive N is shown in blue. Biotin is covalently attached to a lysine residue in the carboxylase enzyme.

Product is acetyl-CoA 2. Malonyl-CoA is not involved no requirement for biotin 3. Oxidative process requires NAD+ and FAD and produces ATP 4. Fatty acids form thioesters with CoA-SH 5. Starts at carboxyl end (CHjCOg") 6. Occurs in the mitochondrial matrix, with no ordered aggregate of enzymes 7. P-Hydroxyacyl intermediates have the L configuration Precursor is acetyl-CoA Malonyl-CoA is source of two-carbon units biotin required Reductive process requires NADPH and ATP Fatty acids form thioesters with acyl carrier proteins (ACP-SH) Starts at methyl end (CHjCHg") Occurs in the cytosol, catalyzed by an ordered multienzyme complex P-Hydroxyacyl intermediates have the D configuration... 

Biotin-dependent enzymes catalyze the decarboxylation of a-keto acids and thioesters. 

There are fewer catalytic ribozymes compared to deoxyribozymes. Examples include a trara-splicing ribozyme, an alcohol dehydrogenase, a ligase capable of functioning at low temperature, " a ribozyme that will ligate the 5 -terminus of RNA to a polypeptide, a transcriptional activator and a tRNA aminoacylation catalyst. An RNA aptamer bearing 5 -CoA has been selected to catalyse thioester formation in the presence of biotin-AMP. In vitro selection has also been used to identify allosteric hairpin ribozymes, activated in the presence of short oligonucleotides, and a ribozyme that catalyses amide bond formation from a 2 -amino nucleotide. " ... 

Biotin was first isolated and identified as a yeast growth factor in 1935. Contrary to the rapid progress made in the field of water soluble vitamins in the 1940s, the function of biotin remained a mystery until 1959 when F. Lynen and his colleagues from Munich observed that bacterial j -methyl-crotonyl-CoA carboxylase carries out the carboxylation of free ( + )-biotin in the absence of its natural CoA-thioester substrate. 

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