A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity

Department of Nutrition & Health Sciences, University of Nebraska-Lincoln, NE 68583-0806, USA.
Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.82). 04/2011; 412(9-10):735-9. DOI: 10.1016/j.cca.2010.12.031
Source: PubMed


Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to both carboxylases and histones. Biotinylated carboxylases and biotinylated histones play crucial roles in the metabolism of fatty acids, amino acids, and glucose, and in gene regulation and genome stability, respectively. HCS null mammals are not viable whereas HCS deficiency is linked to developmental delays in humans and phenotypes such as short life span and low stress resistance in Drosophila.
HCS-dependent biotinylation of the polypeptide p67 was detected and quantified in a 96-well plate format using IRDye-streptavidin and infrared spectroscopy.
Biotinylation of p67 by recombinant HCS (rHCS) and HCS from human cell extracts depended on time, temperature, and substrate concentration, all consistent with enzyme catalysis rather than non-enzymatic biotinylation. The Michaelis-Menten constant of rHCS for p67 was 4.1±1.5 μmol/l. The minimal concentration of rHCS that can be detected by this assay is less than 1.08 nmol/l. Jurkat cells contained 0.14±0.02 U of HCS activity [μmol of biotinylated p67 formed/(nmol/l HCSh)] in 400 μg of total protein.
We developed a 96-well plate assay for high-throughput analysis of HCS activity in biological samples and studies of synthetic and naturally occurring HCS inhibitors.

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    ABSTRACT: Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96 L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.
    European journal of human genetics: EJHG 10/2011; 20(4):428-33. DOI:10.1038/ejhg.2011.198 · 4.35 Impact Factor