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.