Human placental aldose reductase

role of Cys-298 in substrate and inhibitor binding

Aruni Bhatnagar, Si Qi Liu, Norio Ueno, Bireswar Chakrabarti, Satish Srivastava

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Steady-state kinetic and inhibition properties of human placental aldose reductase carboxymethylated at Cys-298 were investigated. A comparison of the primary deuterium kinetic isotope effect on the reduced and the carboxymethylated enzymes suggests that carboxymethylation did not affect the reaction sequence of substrate binding and release. Values of DV/Kd-glyceraldehyde greater than DV suggest that steps in the reaction scheme subsequent to hydride transfer, particularly the release of NADP may be rate limiting. Carboxymethylation of Cys-298 was also found to affect NADPH and aldehyde binding to the enzyme. Carboxymethylation had little effect on the secondary structure of the enzyme, but a comparison of the circular dichroic spectra of the reduced and carboxymethylated enzyme, suggests a weakened interaction between the nicotinamide and 2'-monophosphoadenosine 5'-diphosphoribose of NADPH, and the carboxymethylated enzyme. Interaction between Cys-298 and NADPH appears to determine the rate of isomerization of the E:NADP binary complex and carboxymethylation-induced decrease in kcat may be due to slower isomerization of the E: NADP binary complex. The carboxymethylated enzyme was less sensitive than the reduced enzyme to most aldose reductase inhibitors including sorbinil (d-6-fluoro-spiro[chroman-4,4'-im idazolidine]-2',5'-dione), except tolrestat (N-methyl-N-[(5-trifluromethyl-6-methoxy-1-naphthalenyl)-thiomethyl]glyc ine) and quercetin. On the basis of these observations it is suggested that Cys-298 may form part of the 'S'-inhibitor binding site of the enzyme and may be responsible for tight binding of NADPH.

Original languageEnglish (US)
Pages (from-to)207-214
Number of pages8
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1205
Issue number2
DOIs
StatePublished - Apr 13 1994

Fingerprint

Aldehyde Reductase
NADP
Substrates
Enzymes
Isomerization
Chromans
Glyceraldehyde
Kinetics
Niacinamide
Deuterium
Quercetin
Aldehydes
Hydrides
Isotopes
Binding Sites

Keywords

  • Aldose reductase
  • Circular dichroism
  • Human placenta
  • Isotope effect

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

Cite this

Human placental aldose reductase : role of Cys-298 in substrate and inhibitor binding. / Bhatnagar, Aruni; Liu, Si Qi; Ueno, Norio; Chakrabarti, Bireswar; Srivastava, Satish.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1205, No. 2, 13.04.1994, p. 207-214.

Research output: Contribution to journalArticle

Bhatnagar, Aruni ; Liu, Si Qi ; Ueno, Norio ; Chakrabarti, Bireswar ; Srivastava, Satish. / Human placental aldose reductase : role of Cys-298 in substrate and inhibitor binding. In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular. 1994 ; Vol. 1205, No. 2. pp. 207-214.
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