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, rare congenital deficiency of biotinidase results in severe functional biotin deficiency.

Biotinidase is also the major plasma binding protein for biotin. The pH optimum of the enzyme is 4.5 to 5.5, and its Km 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 excrete large amounts of both biocytin and free biotin. Biotin is co-valently 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)

intermediate in the hydrolysis of biocytin. Free biotin can be released from bi-otinidase at low pH, and the enzyme can also catalyze a biotinoyl transferase reaction, biotinoylating histones, and other nucleophilic acceptors (Hymes and Wolf, 1996, 1999). Biotinidase Deficiency Genetic lack of biotinidase results in the late-onset variant of multiple carboxylase deficiency. Patients generally present later in life than those with holocarboxylase synthetase deficiency (Section and have a lower than normal blood concentration of biotin. Culture of fibroblasts in media containing low concentrations of biotin results in normal activities of carboxylases, and holocarboxylase synthetase activity is normal.

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 reutilization. The delayed development of clinical and biochemical abnormalities is a result of the accumulation of biotin by the fetus, so that at birth the infant has adequate stores of the vitamin.

Biotinidase-deficient patients have higher than normal amounts of biocytin in plasma and urine, and excrete larger than normal amounts of biotin, reflecting the importance of protein binding of biotin to prevent urinary loss. Therapy with 10 mg of biotinper day prevents the development of most symptoms, although some patients develop neurosensory hearing loss and optic atrophy despite therapy with biotin. This reflects the role of biotinidase in tissue uptake of biotin (Section 11.1; Wolf and Feldman, 1982; Wolf and Heard, 1991; Baumgartner and Suormala, 1997, 1999).

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