Bovine lens aldose reductase

pH-dependence of steady-state kinetic parameters and nucleotide binding

Si Qi Liu, Aruni Bhatnagar, Satish Srivastava

Research output: Contribution to journalArticle

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Abstract

The pH-dependence of nucleotide binding and steadystate kinetic parameters of aldehyde reduction and alcohol oxidation catalyzed by bovine lens aldose reductase were studied. The maximal velocity of aldehyde reduction with NADPH and p-chlorobenzaldehyde was pH independent at low pH but decreased at high pH with a pK of 7.6. The V/K of NADPH displayed a bell-shaped dependence on pH and decreased with a pKa of 5.3 and a pKb of 7.5. The dissociation constant of NADPH and 3-acetylpyridine adenine dinucleotide phosphate (3-APADP) increased at low pH with a pK of 5.6-5.8 and at high pH with a pK of 9.4-9.7. The pKi of NADP and NADPH decreased below a pH of 5 and 6.7 and above a pH of 8.5 and 9.7, respectively. The pK of 8.5-9.7 appears to be due to the interaction of the 2′-phosphate of the nucleotide with a protonated base, possibly a lysine residue. The maximal velocity of alcohol oxidation was pH independent at high pH but decreased at low pH with a pK of 6.5-7.0, when p-chlorobenzyl alcohol or benzyl alcohol and 3-APADP were used. The amino acid residue for alcohol binding has a pK of 7.5-8.2 and also appears in pKi profiles of sorbinil, a competitive inhibitor versus the alcohol. Large (3-3.5) isotope effects on maximal velocity obtained with benzyl alcohol and 3-APADP suggest that with these substrates the hydride transfer step is rate-limiting and a pK of 6.5-7.0 may be the true pK of the acid-base catalyst, possibly a histidine.

Original languageEnglish (US)
Pages (from-to)25494-25499
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number34
StatePublished - Dec 5 1993

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Aldehyde Reductase
NADP
Kinetic parameters
Lenses
Nucleotides
Alcohols
Benzyl Alcohol
Aldehydes
Oxidation
Histidine
Hydrides
Isotopes
Lysine
Phosphates
Amino Acids
Catalysts
Acids
Substrates
3-acetylpyridine-adenine dinucleotide phosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bovine lens aldose reductase : pH-dependence of steady-state kinetic parameters and nucleotide binding. / Liu, Si Qi; Bhatnagar, Aruni; Srivastava, Satish.

In: Journal of Biological Chemistry, Vol. 268, No. 34, 05.12.1993, p. 25494-25499.

Research output: Contribution to journalArticle

Liu, Si Qi ; Bhatnagar, Aruni ; Srivastava, Satish. / Bovine lens aldose reductase : pH-dependence of steady-state kinetic parameters and nucleotide binding. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 34. pp. 25494-25499.
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