Biotin-Containing Enzymes

Hilbi H, R Hermann, P Dimroth (1993) The malonate decarboxylase enzyme system of Malonomonas rubra evidence for the cytoplasmic location of the biotin-containing component. Arch Microbiol 160 126-131. 

Biocytin is e-N-biotinyl-L-lysine, a derivative of D-biotin containing a lysine group coupled at its e-amino side chain to the valeric acid carboxylate. It is a naturally occurring complex of biotin that is typically found in serum and urine, and probably represents breakdown products of recycling biotinylated proteins. The enzyme biotinidase specifically cleaves the lysine residue and releases the biotin component from biocytin (Ebrahim and Dakshinamurti, 1986, 1987). 

Purify the SH-labeled oligo by gel filtration on a desalting resin using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The probe now may be used to conjugate with an activated enzyme, biotin, fluorescent tag, or other molecules containing a sulfhydryl-reactive group. 

Biotin-Containing Enzymes. 2. The Mechanism of Biotin Action 738 Box 14-C The Vitamin B6 Family Pyridoxine, Pyridoxal, and Pyridoxamine... 

Biotin contains three chiral centers and therefore has eight stereoisomers.1819 Of these, only one, the dextrorotatory (-i-)-biotin, is biologically active.19 20 The vitamin is readily oxidized to die sulfoxide and sul-fone. The sulfoxide can be reduced back to biotin by a molybdenum-containing reductase in some bacteria (see also Chapter 16, Section H).20a Biotin is synthesized from pimeloyl-CoA (see chapter banner, p. 719 and Eq. 24-39). Four enzymes are required. Two of them, a... 

The pathway The first committed step in fatty acid biosynthesis is the carboxylation of acetyl CoA to form malonyl CoA which is catalyzed by the biotin-containing enzyme acetyl CoA carboxylase. Acetyl CoA and malonyl CoA are then converted into their ACP derivatives. The elongation cycle in fatty acid synthesis involves four reactions condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP releasing free ACP and C02, then reduction by NADPH to form D-3-hydroxybutyryl-ACP, followed by dehydration to crotonyl-ACP, and finally reduction by NADPH to form butyryl-ACP. Further rounds of elongation add more two-carbon units from malonyl-ACP on to the growing hydrocarbon chain, until the C16 palmitate is formed. Further elongation of fatty acids takes place on the cytosolic surface of the smooth endoplasmic reticulum (SER). 

Avidin inhibits carbamyl-P synthesis by rat liver enzyme (10 units cause a 40 to 70% inhibition, depending on the purity of the enzyme), but does not affect the frog liver preparations. Preincubation of the avidin with a large excess of biotin does not prevent the inhibition of the mammalian enzyme, suggesting that the effect is not strictly due to the biotin-binding capacity of avidin. We concluded that biotin is not directly involved in the synthesis of carbamyl-P, although the enzyme may contain bound biotin. Also, this vitamin may play a role in enzyme synthesis (34). 

Methane and carbon dioxide are the exceptions. Methane is an end product of anaerobic metabolism of many microorganisms, and carbon dioxide (for carboxylation) is handled by biotin-containing enzymes. 

To examine the purity of the protein that was eluted from a Sephacryl S-300 column, we subjected the protein to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and then performed a Western blot (26) using avidin linked to phosphatase as a probe. The SDS-PAGE indicated that there were many polypeptides in this partly purified preparation (Fig. 6). Among these were two major and several minor biotin-containing bands (Fig. 7). This preparation may contain propionyl CoA carboxylase or a partly degraded form of ACCase that can use propionyl CoA as well as acetyl CoA as a substrate. Therefore, the enzyme data presented here are quali-... 

Avidin Biotin-containing enzymes and biotinylated enzymes... 

Dietary deficiency of biotin sufficient to cause clinical signs is extremely rare in human beings, although it may be a problem in intensively reared poultry. However, there is increasing evidence that suboptimal biotin status may be relatively common, despite the fact that the vitamin is widely distributed in many foods, is synthesized by intestinal flora, and there is an efficient mechanism for conserving biotin after the catabolism of biotin-containing enzymes. 

As a result of this resorption and the protein binding of plasma biotin, which reduces filtration at the glomerulus, renal clearance of biotin is only 40% of that of creatinine. This efficient conservation of biotin, together with the recycling of biocytin released from the catabolism of biotin-containing enzymes, may be as important as intestinal bacterial synthesis of the vitamin in explaining the rarity of deficiency. 

Proteolysis of biotin-containing enzymes releases biocytin, either as free biotinyl-lysine or as a variety of small biocytin-containing peptides the e-amino lysine link of biocytin is not a substrate for peptidases. 

Biocytin is hydrolyzed by biotinidase, which acts on free or peptide-incorporated biocytin to release biotin, but has no general peptidase or esterase activity. Biotinidase is most active toward free biocytin, but it will also release biotin from biocytin-containing peptides. The activity decreases as the size of the peptide increases, so it is likely that in vivo the catabolism of biotin-containing enzymes is by proteolysis, followed by biotinidase action, rather than the release of biotin, leaving the apoenzyme as a substrate for proteolysis. Biotinidase is found in all tissues, including the pancreatic juice and intestinal mucosa. 

Biotinidase functions both to release free biotin from biocytin in foods, and to recycle and conserve biotin after turnover of biotin-containing enzymes. As discussed in Section 11.2.3.1, rare congenital deficiency of biotinidase results in severe functional biotin deficiency. 

The problem is a functional deficiency of biotin, due both to inability to release free biotin from dietary biocytin and also to failure of the normal recovery of free biotin by biotinidase action on the biocytin released by proteolysis of biotin-containing enzymes. Normal intakes of biotin are inadequate to meet the requirements of these patients the provision of pharmacological doses of free biotin provides an adequate amount to meet requirements without the need for reutUization. The delayed development of clinical and biocheimcal abnormalities is a result of the accumulation of biotin by the fetus, so that at birth the infant has adequate stores of the vitamin. 

Vol. 62 [57]. Antibodies that Bind Biotin and Inhibit Biotin-Containing Enzymes. M. Berger. Vol. 66 [102]. Preparation of an Antiserum to Sheep Liver Dihydropteridine Reductase. S. Milstein and S. Kaufman. 

---