Biotin-dependent enzymes

FIGURE 18.32 Biotin is covalently linked to a protein via the e-amino group of a lysine residue. The biotin ring is thus tethered to the protein by a 10-atom chain. It functions by carrying carboxyl groups between distant sites on biotin-dependent enzymes. 

Knowle.s, J. R., 1989. The mechani.sm of biotin-dependent enzyme.s. Annual Review of Biochemistry 58 195—221. 

Fatty acids with odd numbers of carbon atoms are rare in mammals, but fairly common in plants and marine organisms. Humans and animals whose diets include these food sources metabolize odd-carbon fatty acids via the /3-oxida-tion pathway. The final product of /3-oxidation in this case is the 3-carbon pro-pionyl-CoA instead of acetyl-CoA. Three specialized enzymes then carry out the reactions that convert propionyl-CoA to succinyl-CoA, a TCA cycle intermediate. (Because propionyl-CoA is a degradation product of methionine, valine, and isoleucine, this sequence of reactions is also important in amino acid catabolism, as we shall see in Chapter 26.) The pathway involves an initial carboxylation at the a-carbon of propionyl-CoA to produce D-methylmalonyl-CoA (Figure 24.19). The reaction is catalyzed by a biotin-dependent enzyme, propionyl-CoA carboxylase. The mechanism involves ATP-driven carboxylation of biotin at Nj, followed by nucleophilic attack by the a-carbanion of propi-onyl-CoA in a stereo-specific manner. 

This biotin-dependent enzyme [EC 6.4.1.5] catalyzes the following reaction ATP -i- geranoyl-CoA -i- HCOs = ADP + Pi + 3-(4-methylpent-3-en-l-yl)pent-2-enedi-oyl-CoA. 

This biotin-dependent enzyme [EC 6.4.1.4] catalyzes the reaction of ATP with 3-methylcrotonyl-CoA and bicarbonate to produce ADP, orthophosphate, and 3-methyl-glutaconyl-CoA. 

BIQCHEMICAL NQMENCATURE BIOTIN AND DERIVATIVES Biotin-dependent enzymes,... 

Knowles, J. (1989) The mechanism of biotin-dependent enzymes. Annu. Rev. Biochem. 58, 195-221. 

A. Structure of biotin. B. Biotin covalently bound to a lysyl residue of a biotin-dependent enzyme. 

When pyruvate with a chiral methyl group is carboxylated by pyruvate carboxylase the configuration at C-3 is retained. The carboxyl enters from the 2-si side, the same side from which the proton (marked H ) was removed to form the enolate anion (Eq. 14-12). Comparable stereochemistry has been established for other biotin-dependent enzymes.64 65... 

As discussed in Section 11.2.2, biotin is incorporated covalently into biotin-dependent enzymes as the e-amino-lysine peptide, biocytin. On catabolism... 

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. 

Steady-state kinetic analysis shows that biotin-dependent reactions proceed by way of a two-site ping-pong mechanism the two-part reactions are catalyzed at distinct sites in the enzyme. These sites may be on the same or different polypeptide chains in different biotin-dependent enzymes. The e-amino linkage of lysine to the side chain of biotin in biocytin allows considerable movement of the coenzyme - the distance from C-2 of lysine to C-5 of biotin is IdA, thus allowing movement of biotin between the carboxylation and carboxyltransfer sites. 

Holocarboxylase synthetase from a wide variety of species wUl act on all four apocarboxylases from other species and on a variety of bacterial biotin-dependent apoenzymes. In all the biotin-dependent enzymes investigated to date, the reactive lysine residue is flanked by methionine residues on both sides, and there is a high degree of conservation of the amino sequence around this Met-Lys-Met sequence (Chapman-Smith and Cronan, 1999a, 1999b). 

How is oxaloacetate replenished Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate. Rather, oxaloacetate is formed by the carboxylation of pyruvate, in a reaction catalyzed by the biotin-dependent enzyme pyruvate carboxylase. 

Chemical and catalytic mechanisms of carboxyl transfer in biotin-dependent enzymes 02ACR113. 

The metabolic action of biotin is mediated through biotin-dependent enzymes that actively synthesize purines. It is a stable monocarboxylic acid, soluble in water and alcohol, and acts as a coenzyme as well as a growth factor, even in very small quantities. Biotin deficiencies cause scaly dermatitis, hyperkeratosis and alopecia. Topical application of biotin reduces the secretion of sebum. 

Acetyl-CoA carboxylase is a biotin-dependent enzyme. It has been purified from microorganisms, yeast, plants, and animals. In animal cells, it exists as an inactive pro-tomer (M.W. 400,000) and as an active polymer (M.W. 4-8 million). The protomer contains the activity of biotin carboxylase, biotin carboxyl carrier protein (BCCP), transcarboxylase, and a regulatory allosteric site. Each protomer contains a biotinyl group bound in amide linkage to the e-amino group of a lysyl residue. 

Other biotin-dependent enzymes include propionyl-CoA carboxylase and pyruvate carboxylase (Chapter 15). The latter, like acetyl-CoA carboxylase, is subject to allosteric regulation. Pyruvate carboxylase, a mitochondrial enzyme, is activated by acetyl-CoA and converts pyruvate to oxaloacetate which, in turn, is converted to glucose via... 

In the homogeneous assay, an enzyme modulator (antibody, inhibitor or receptor for the enzyme) is covalently linked to the ligand which competes with free ligand from the test sample for the available antibodies. The activity of the indicator enzyme depends on the amount of free modulator. As the amount of hapten increases, less hapten-modulator is bound by the antibody and the activity of the indicator enzyme (which is not conjugated) is more modulated (inhibited, e.g., avidin for biotin-dependent enzymes (Section 14.3.1.2), or activated, e.g., antibody to wild-type BGase with an inactive mutant of this enzyme. Section 10.2.2.1). 

The answer is b. (Murray, pp 627-661. Scriver, pp 3897-3964. Sack, pp 121-138. Wilson, pp 287-320.) The vitamin biotin is the cofactor required by carboxylating enzymes such as acetyl CoA, pyruvate, and propionyl CoA carboxylases. The fixation of CO2 by these biotin-dependent enzymes occurs in two stages. In the first, bicarbonate ion reacts with adenosine triphosphate (ATP) and the biotin carrier protein moiety of the enzyme in the second, the active CO2 reacts with the substrate—e.g., acetyl CoA. 

The formation of a bond between the carboxylate group derived from bicarbonate and a carbon adjacent to a carbonyl group is indicative of a reaction catalyzed by an enzyme that utilizes biotin as a cofactor (Scheme 16). The recent review by Knowles covers many recent discoveries relating to the role of enzymes in these reactions (43). (Editor s note For additional aspects of biotin-dependent carboxylation, see Chapter 6 by O Leary.) Most biotin-dependent enzymes promote a two-step process in which Ai-carboxybiotin serves as an intermediate in a process involving the exchange of the caiix)xylate group derived from bicarbonate for a proton at the a-carbon of the carbonyl compound (44). 

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