Deficiency of Biotinyl-AMP Synthetase Activity in Fibroblasts of Patients with Holocarboxylase Synthetase Deficiency

A simple, rapid assay was developed to diagnose holocarboxylase synthetase deficiency. Holocarboxylase synthetase first catalyzes the formation of biotinyl-AMP from biotin and ATP, an activity designated as biotinyl-AMP synthetase. In the second step of the reaction, biotin is transferred from biotinyl-AMP to the enzymatically inactive apocarboxylase to form an active holocarboxylase. The assay for holocarboxylase synthetase activity therefore requires a protein apocarboxylase substrate which is not readily available. In the assay for biotinyl-AMP synthetase, hydroxylamine reacts nonenzymatically with the product of the enzymatic reaction, biotinyl-AMP, to form biotinylhydroxamate. At the end of the reaction, unreacted radioactive biotin substrate, which is negatively charged at neutral pH, is bound to an anion-exchange resin and a neutral radioactive biotinylhydroxamate product in the supernatant is counted. In fibroblasts from 11 patients with proven holocarboxylase synthetase deficiency, the mean biotinyl-AMP synthetase activity at 25 nM biotin was 4% of the control mean with a range of 0.2 to 8%. This is an improved assay because it does not require preparation of an apocarboxylase substrate and is suitable for the diagnosis of patients with holocarboxylase synthetase deficiency.