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Expression of human aldose and aldehyde reductases. Site-directed mutagenesis of a critical lysine 262

K M Bohren1, J L Page, R Shankar

  • 1Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.

The Journal of Biological Chemistry
|December 15, 1991
PubMed

Insights

Lysine 262 is essential for human aldose reductase and aldehyde reductase activity, impacting cofactor binding. Mutating this residue significantly reduces enzyme function, offering insights into diabetic complication therapies.

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Human aldose reductase (hAR) and aldehyde reductase (hGR) are implicated in diabetic complications.
  • Inhibiting these enzymes is a therapeutic strategy for diabetic complications.

Purpose of the Study:

  • To investigate the role of lysine 262 in hAR and hGR structure and function.
  • To understand the impact of lysine 262 mutations on enzyme kinetics and cofactor binding.

Main Methods:

  • Site-directed mutagenesis was used to replace lysine 262 with methionine in hAR and hGR cDNAs.
  • Wild-type and mutant enzymes were overexpressed in E. coli and purified.
  • Kinetic studies (Km values) were performed to assess enzyme activity and cofactor binding.

Main Results:

  • Mutant hAR (K262M) showed a 66-fold increase in Km for NADPH and a 35-fold increase for DL-glyceraldehyde.
  • Mutant hGR (K262M) exhibited a 97-fold increase in Km for NADPH and an 86-fold increase for DL-glyceraldehyde.
  • These changes indicate lysine 262 is critical for cofactor binding and catalytic activity.

Conclusions:

  • Lysine 262 is crucial for the catalytic activity of both human aldose reductase and aldehyde reductase.
  • The mutation at lysine 262 significantly impairs cofactor binding, affecting enzyme function.
  • Understanding this residue's role can inform the development of targeted inhibitors for diabetic complications.

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