Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303

J. M. Petrash, T. M. Harter, C. S. Devine, P. O. Olins, A. Bhatnagar, S. Liu, S. K. Srivastava

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Abstract

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys-298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'(m(NADPH)), K'(m(DL-glyceraldehyde)), and k(cat(DL-glyceraldehyde)) relative to wild type aldose reductase as well as a 10-fold higher K(i) for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in k(cat) as well as an increase in K'(m(DL-glyceraldehyde)) and K(i(sorbinil)). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.

Original languageEnglish (US)
Pages (from-to)24833-24840
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number34
StatePublished - Jan 1 1992

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Petrash, J. M., Harter, T. M., Devine, C. S., Olins, P. O., Bhatnagar, A., Liu, S., & Srivastava, S. K. (1992). Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303. Journal of Biological Chemistry, 267(34), 24833-24840.