Biotin enzyme

To clarify the characteristics of AMDase, the effects of some additives were examined using phenylmalonic acid as the representative substrate. The addihon of ATP and coenzyme A did not enhance the rate of the reaction, different from the case of malonyl-CoA decarboxylase and others in those, ATP and substrate acid form a mixed anhydride, which in turn reacts with coenzyme A to form a thiol ester of the substrate. In the present case, as both ATP and CoA-SH had no effect, the mechanism of the reaction will be totally different from the ordinary one described above. It is well estabhshed that avidin is a potent inhibitor of the formation of the biotin-enzyme complex. In the case of AMDase, addition of avidin has no influence on the enzyme activity, indicating that AMDase is not a biotin enzyme. 

The reaction is catalyzed by the biotin,enzyme acetyl-CoA carboxylase (E-biotin) assisted by Mg2+ ions. This enzyme is a tetramer with a molecular mass of 400 000-500000. 

The nucleophile in biological Claisen reactions that effectively adds on acetyl-CoA is almost always malonyl-CoA. This is synthesized from acetyl-CoA by a reaction that utilizes a biotin-enzyme complex to incorporate carbon dioxide into the molecule (see Section 15.9). This has now flanked the a-protons with two carbonyl groups, and increases their acidity. The enzymic Claisen reaction now proceeds, but, during the reaction, the added carboxyl is lost as carbon dioxide. Having done its job, it is immediately removed. In contrast to the chemical analogy, a carboxylated intermediate is not formed. Mechanistically, one could perhaps write a concerted decarboxylation-nucleophilic attack, as shown. An alternative rationalization is that decarboxylation of the malonyl ester is used by the enzyme to effectively generate the acetyl enolate anion without the requirement for a strong base. 

This mixed anhydride carboxylates the coenzyme in a biotin-enzyme complex. Biotin is bound to a lysine residue in the enzyme as an amide. The carboxylation... 

In what can be considered a reversal of this sequence, the acetyl-CoA acts as the nueleophile and is carboxylated to malonyl-CoA with displacement of the biotin-enzyme system. 

Fixation of carbon dioxide by biotin-enzyme complexes is not unique to acetyl-CoA, and another important example occurs in the generation of oxaloacetate from pyravate in the synthesis of glucose from non-carbohydrate sources (gluconeogene-sis). This reaction also allows replenishment of Krebs... 

Biotin — Enzyme-bound biotin Carboxylation reactions i... 

Biotin becomes attached to the proper e-amino groups at the active centers of biotin enzymes by the action of biotin holoenzyme synthetase (biotinyl... 

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 ... 

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. 

Thiamine, biotin and pyridoxine (vitamin B) coenzymes are grouped together because they catalyze similar phenomena, i.e., the removal of a carboxyl group, COOH, from a metabolite. However, each requires different specific circumstances. Thiamine coenzyme decarboxylates only alpha-keto acids, is frequently accompanied by dehydrogenation, and is mainly associated with carbohydrate metabolism. Biotin enzymes do not require the alpha-keto configuration, are readily reversible, and are concerned primarily with lipid metabolism. Pyridoxine coenzymes perform nonoxidative decarboxylation and are closely allied with amino acid metabolism. 

Transfer membrane to fresh plastic bag containing avidin-biotin-enzyme solution. Incubate 30 min at room temperature with slow rocking, then wash over a 30-min span as in step 4. 

Hybridization in a plastic bag requires 5 to 10 ml avidin-biotin-enzyme solution. Membrane strips require 5 to 10 ml/strip, whereas blots from standard-sized gels (i.e., 14 x 16 cm) require 50 ml for convenient handling in a tray. 

Claisen reactions involving acetyl-CoA are made even more favourable by first converting acetyl-CoA into malonyl-CoA by a carboxylation reaction with CO2 using ATP and the coenzyme biotin (Figure 2.9). ATP and CO2 (as bicarbonate, HC03-) form the mixed anhydride, which car-boxy lates the coenzyme in a biotin-enzyme complex. Fixation of carbon dioxide by biotin-enzyme complexes is not unique to acetyl-CoA, and another important example occurs in the generation of oxaloacetate from pyruvate in the synthesis of glucose from non-carbohydrate sources... 

Samols D, Thornton CG, Murtif VL et al. Evolutionary conservation among biotin enzymes. J Biol Chem 1988 263 6461. 

Acetyl-CoA is a positive effector of pyruvate carboxylase, a biotin enzyme. The correct answer is 1,3-diphosphoglyceric acid. It is produced by the oxidation of glyceraldehyde-3-phosphate in the presence of P,. 

The original interest in avidin was because of the egg white injury that was subsequently shown to be avidin-induced biotin deficiency. Thereafter, avidin was used because of its high affinity for biotin (a dissociation constant of 10 mol per L), not only to induce experimental biotin deficiency, but also to bind to biotin in isolated enzymes and thus, by irreversible inhibition, demonstrate the coenzyme role of biotin. Because of the stability of the avidin-biotin complex, it has not been possible to use immobilized avidin as a means of purifying biotin enzymes - there seems to be no way in which the enzyme can be released from avidin binding. Because of its high affinity for biotin, avidin is used to provide an extremely sensitive system for linking reporter molecules in a variety of analytical systems. 

Dakshinamurti K and Chauhan J (1988) Regulation of biotin enzymes. Annual Reviews of Nutrition 8,211-33. 

CO -biotin-enzyme + pyruvate. - biotin-enzyme + oxaloacetate... 

Biotin snatcher. Avidin, a 70-kd protein in egg white, has very high affinity for biotin. In fact, it is a highly specific inhibitor of biotin enzymes. Which of the following conversions would be blocked by the addition of avidin to a cell homogenate ... 

ATP to yield the d isomer of methylmalonyl CoA (Figure 22.11). This carboxylation reaction is catalyzed by propionyl CoA carboxylase, a biotin enzyme that is homologous to and has a catalytic mechanism like that of pyruvate carboxylase (Section 16.3.2). The d isomer of methylmalonyl CoA is racemized to the 1 isomer, the substrate for a mutase that converts it into succinyl CoA by an intramolecular rearrangement. The -CO-S-CoA group migrates from C-2 to C-3 in exchange for a hydrogen atom. This very unusual isomerization is catalyzed by methylmalonyl CoA mutase, which contains a derivative of vitamin Bj2, cobalamin, as its coenzyme. 

CO2-biotin-enzyme + acetyl Co A —> malonylCoA -I- biotin-enzyme This enzyme is also the essential regulatory enzyme for fatty acid metabolism (Section 22,5). 

Conversion of acetyl-CoA to malonyl-CoA with a biotin enzyme (acetyl-CoA... 

Arinze, J. C., and Mistry, S. P (1971). Activities of some biotin enzymes and certain aspects of gluconeogenesis during biotin deficiency. Comp. Biochem. PJjysEoL B 38,285-294. 

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