Holocarboxylase synthetase (HCS) is an essential enzyme for the biotinylation of several mammalian carboxylases. A deficiency of HCS is accountable for early onset biotin-responsive multiple carboxylase deficiency. To address the mechanism of biotin responsiveness, we analyzed the kinetic properties of the previously identified mutant. L237P, and another mutant, V550M, described in this report. The V550M mutant contains a G to A transition at position 1935, which is within the putative biotin binding site, whereas the mutation in L237P occurs outside the biotin binding site.KmandVmaxvalues for the mutant proteins were determined by overexpressing cDNAs encoding the mutants in transformed fibroblasts from an HCS-deficient patient. Enzyme activity assays were performed using apocarboxyl carrier protein as a substrate. AKmfor biotin that was larger than the value found for the wild-type cDNA was observed in fibroblasts transfected with the V550M cDNA, but not the L237P cDNA. TheVmaxfor the expressed L237P cDNA was 4.3% of that observed for the wild-type cDNA. Biotin-responsiveness in the patient with the L237P mutation was neither due to an increased affinity for biotin nor a restoration of stability of the mutant by biotin treatment. A new mechanism of biotin responsiveness in HCS deficiency is presented.