CHARACTERIZATION OF MUTANT HOLOCARBOXYLASE SYNTHETASE (HCS) - A K-M FOR BIOTIN WAS NOT ELEVATED IN A PATIENT WITH HCS DEFICIENCY

Holocarboxylase synthetase (HCS) is an essential enzyme for the biotin ylation of several mammalian carboxylases. A deficiency of HCS is acco untable for early onset biotin-responsive multiple carboxylase deficie ncy. To address the mechanism of biotin responsiveness, we analyzed th e kinetic properties of the previously identified mutant, L237P, and a nother mutant, V550M, described in this report. The V550M mutant conta ins 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. K-m and V-max values for the mutant proteins wer e determined by overexpressing cDNAs encoding the mutants in transform ed fibroblasts from an HCS-deficient patient. Enzyme activity assays w ere performed using apocarboxyl carrier protein as a substrate. A K-m for 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. The V-max for the expressed L237P cDNA was 4.3% of tha t observed for the wild-type cDNA. Biotin-responsiveness in the patien t with the L237P mutation was neither due to an increased affinity for biotin nor a restoration of stability of the mutant by biotin treatme nt. A new mechanism of biotin responsiveness in HCS deficiency is pres ented.