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Related Experiment Videos

Human serum biotinidase is a thiol-type enzyme.

K Hayakawa1, J Oizumi

  • 1Division of Metabolism, National Children's Medical Research Center, Tokyo.

Journal of Biochemistry
|May 1, 1988
PubMed
Summary

This study reveals that sulfhydryl groups are essential for biotinidase activity. While reducing agents enhance activity, blocking agents inhibit it, indicating a crucial role for these residues in enzyme function.

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Area of Science:

  • Biochemistry
  • Enzymology

Background:

  • Biotinidase activity is crucial for biotin metabolism.
  • Understanding the role of sulfhydryl groups is key to enzyme function.

Purpose of the Study:

  • To investigate the effects of reducing and blocking agents on human serum and purified biotinidase activity.
  • To elucidate the role of sulfhydryl residues in biotinidase function.

Main Methods:

  • Development of a novel High-Performance Liquid Chromatography (HPLC) assay to directly measure p-aminobenzoate (PAB).
  • Testing the effects of 2-mercaptoethanol (ME), organic mercurials, N-ethylmaleimide (NEM), and dithiobis(2-nitro)benzoic acid (DTNB) on enzyme activity.
  • Quantification of sulfhydryl groups using Ellman's assay.

Main Results:

  • 2-mercaptoethanol (ME) enhanced biotinidase activity in human sera (n=83) by 100-400% at 1 mM.
  • Organic mercurials and alkylating agents (NEM, DTNB) inhibited purified biotinidase activity.
  • Low concentrations of mercurials and mercuric ions showed slight enhancement (20-30%) of purified enzyme activity.
  • Serine protease inhibitors (PMSF, DFP) did not inhibit biotinidase activity.

Conclusions:

  • The results strongly suggest the presence of an essential sulfhydryl residue at the active center of biotinidase.
  • Biotinidase contains approximately 2.5 sulfhydryl groups per molecule.
  • The enzyme's activity is modulated by the redox state of its sulfhydryl groups.

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