Characterization of Mutant Holocarboxylase Synthetase (HCS): AKm for Biotin Was Not Elevated in a Patient with HCS Deficiency

Abstract

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. Km and Vmax values 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 apo-carboxyl carrier protein as a substrate. A Km 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 Vmax for 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.

Mutations in the holocarboxylase synthetase geneHLCS

“…Two Italian patients and one African patient were also reported to have the V550M mutation [Hwu et al, 2000;Morrone et al, 2002;Tang et al, 2003]. The mutation resulted in a decreased affinity of HLCS to biotin (i.e., a Km mutant); the mutant HLCS shows its enzymatic activity to some extent at high biotin concentrations [Aoki et al, 1997]. The R508W mutant alleles in Japanese patients were associated with either haplotype 2-3 or haplotype 1-4 .…”

“…Another mutation found in several ethnic groups is V550M. One Japanese patient was homozygous for this mutation [Aoki et al, 1997]. Dupuis et al [1996] reported that three U.S. patients had this mutation (four alleles).…”

Haplotype analysis suggests that the two predominant mutations in Japanese patients with holocarboxylase synthetase deficiency are founder mutations

“…The 508Arg Ͼ Trp (1809C Ͼ T) mutation has been found in two unrelated Japanese patients (Sakamoto et al 1998), as well as in non-Japanese patients (Dupuis et al 1996). The 550Val Ͼ Met (1935G Ͼ A) mutation has also been found in many ethnic groups (Dupuis et al 1996;Aoki et al 1997;Zammarchi et al 1998). To determine the origin of these mutations in families with an HCS deficiency, we analyzed the haplotypes of normal and mutant HCS alleles.…”

“…The haplotype of the Japanese patient was 1-4/1-4, whereas that of the Jewish patient was 2-3/2-3. The nucleotide change in the 508Arg Ͼ Trp and the 550Val Ͼ Met mutations is a C-to-T transition at the CpG dinucleotide, and is found in several ethnic groups (Dupuis et al 1996;Aoki et al 1997;Zammarchi et al 1998). These data are in accord with the notion that 508Arg Ͼ Trp and 550Val Ͼ Trp represent hotspot mutations.…”

“…The 997T Ͼ C (237Leu Ͼ Pro) and the 1067delG mutations were screened, using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP), as described (Aoki et al 1995). The 508Arg Ͼ Trp (1809C Ͼ a Underlined haplotypes represent mutant alleles T), 565Arg Ͼ Ter (1980C Ͼ T) and 550Val Ͼ Met (1935G Ͼ A) mutations were determined by direct sequencing, as described (Aoki et al 1997;Sakamoto et al 1998). …”