Enzymatic diagnosis of holocarboxylase synthetase deficiency using apo-carboxyl carrier protein as a substrate

We developed a simple and sensitive method for assessing holocarboxylase synthetase (HCS) activity that is based on measuring incorporation of [³H]biotin into apo-carboxyl carrier protein, a subunit of acetyl-CoA carboxylase from E. coli. Kinetic analysis of HCS from normal fibroblasts showed that the K(m) for biotin was 260 ± 94 nmol/l (mean ± S.D.; n = 5). In contrast, the K(m) values of HCS from two cell lines derived from patients with HCS deficiency were 7200 and 3700, clearly distinguishable from the control value. The sensitivity of this assay was so high that we were able to characterize a mutant enzyme whose activity had not been previously detected. Our method is useful for enzymatic diagnosis of HCS deficiency and characterization of HCS.