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Journal of Biosciences

, Volume 8, Issue 1–2, pp 107–119 | Cite as

Biophysical investigations on the active site of brain hexokinase

  • U. W. Kenkare
  • G. K. Jarori
  • S. R. Kasturi
  • A. Mehta
  • M. P. Pitale
Article

Abstract

Replacement of Mg (II), the natural activator of brain hexokinase (EC 2.7.1.1) by paramagnetic Mn (II) without affecting the physiological properties of the enzyme, has rendered brain hexokinase accessible to investigations by magnetic resonance methods. Based on such studies, a site on the enzyme, where Mn (II) binds directly with high affinity has been identified and characterized in detail. Use ofβ,γ-bidentate Cr (III) ATP as an exchange-inert analogue for Mn (II) ATP has shown that Mn (II) binding directly to the enzyme has no catalytic role but another Mn (II) ion binding simultaneously and independently to the enzyme through the nucleotide bridge participates in enzyme function. However, using this direct binding Mn (II) ion and a covalently bound spin label as paramagnetic probes a beginning has been made in mapping the ligand binding sites of the enzyme. Ultra-violet difference spectroscopy has revealed the presence of at least two glucose 6-phosphate locations on the enzyme one of which presumably is the high affinity regulatory site modulated by substrate glucose. Elution behaviour of the enzyme on a phosphocellulose column suggests that glucose induces a specific phosphate site on the enzyme to which the phosphate bearing regulatory ligands of the enzyme may bind.

Keywords

Hexokinase brain active site magnetic resonance studies enzyme ligand interactions ligand site mapping 

Abbreviations used

Glc-6-P

Glucose 6-phosphate

Pi

inorganic phosphate

PRR

proton relaxation rate

PRE

proton relaxation rate enhancement

NEM

N-ethyl morpholine

DTNB

5, 5’-dithiobis-(2-nitro-benzoic acid)

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Copyright information

© Indian Academy of Sciences 1985

Authors and Affiliations

  • U. W. Kenkare
    • 1
  • G. K. Jarori
    • 1
  • S. R. Kasturi
    • 1
  • A. Mehta
    • 1
  • M. P. Pitale
    • 1
  1. 1.Tata Institute of Fundamental ResearchBombayIndia

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