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Related Experiment Videos

Structure and functional relationships in human pur H

G P Beardsley1, E A Rayl, K Gunn

  • 1Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8064, USA.

Advances in Experimental Medicine and Biology
|May 23, 1998
PubMed
Summary
This summary is machine-generated.

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The human purine synthesis gene (ATIC) was cloned and characterized. Structural and kinetic studies revealed its bifunctional enzyme

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • The human pur H (ATIC) gene encodes the bifunctional enzyme hPurH, crucial for purine nucleotide synthesis.
  • hPurH possesses both AICARFT and IMPCH enzymatic activities, essential for the final steps of purine biosynthesis.
  • Understanding hPurH function is vital for comprehending purine metabolism and related disorders.

Purpose of the Study:

  • To clone, sequence, and characterize the human pur H (ATIC) gene and its protein product, hPurH.
  • To elucidate the structural and functional properties of hPurH, including its catalytic mechanisms and substrate binding.
  • To identify key amino acid residues involved in hPurH catalysis and substrate interaction.

Main Methods:

  • Gene cloning, sequencing, and overexpression in E. coli.

Related Experiment Videos

  • Protein purification, crystallization, and X-ray crystallography for structural determination.
  • Enzymatic assays, kinetic inhibition studies (Cleland-type), and equilibrium sedimentation.
  • Truncation mutant studies to identify functional domains.

    • Main Results:

    • The human pur H gene (ATIC) was cloned, sequenced, and localized to chromosome 2q34-q35.
    • hPurH exists as a dimer in its crystal structure, with a unique cofactor binding site formed by both subunits.
    • Kinetic studies indicate an ordered, sequential reaction mechanism for AICARFT activity, with a rate-limiting late step.
    • Specific residues, Lysine 265 and Histidine 266, were identified as critical for AICARFT activity.

    Conclusions:

    • The study provides comprehensive structural and functional insights into the human bifunctional enzyme hPurH (ATIC).
    • The dimeric structure and identified active site residues offer a basis for understanding purine synthesis regulation.
    • Further investigation is warranted to precisely define the roles of identified residues in catalysis versus substrate binding.