Human liver aldehyde reductase

pH dependence of steady-state kinetic parameters

Aruni Bhatnagar, Ballabh Das, Si Qi Liu, Satish Srivastava

Research output: Contribution to journalArticle

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Abstract

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2′-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2′-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V K for NADPH, the V K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2′-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V K for dl-glyceraldehyde and V K for l-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as d-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V K for 3-APADPH, and V K for d-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.

Original languageEnglish (US)
Pages (from-to)329-336
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume287
Issue number2
DOIs
StatePublished - 1991

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Aldehyde Reductase
NADP
Kinetic parameters
Liver
Phosphates
Adenine
Aldehydes
Glucuronic Acid
Ribose
Nucleotides
Coenzymes
Alcohols
Enzymes
Isotopes
Oxidation
Adenosine Triphosphate
Glyceraldehyde
Deprotonation
Deuterium
Protonation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Human liver aldehyde reductase : pH dependence of steady-state kinetic parameters. / Bhatnagar, Aruni; Das, Ballabh; Liu, Si Qi; Srivastava, Satish.

In: Archives of Biochemistry and Biophysics, Vol. 287, No. 2, 1991, p. 329-336.

Research output: Contribution to journalArticle

Bhatnagar, Aruni ; Das, Ballabh ; Liu, Si Qi ; Srivastava, Satish. / Human liver aldehyde reductase : pH dependence of steady-state kinetic parameters. In: Archives of Biochemistry and Biophysics. 1991 ; Vol. 287, No. 2. pp. 329-336.
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abstract = "The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2′-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2′-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V K for NADPH, the V K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2′-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V K for dl-glyceraldehyde and V K for l-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as d-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V K for 3-APADPH, and V K for d-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.",
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N2 - The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2′-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2′-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V K for NADPH, the V K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2′-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V K for dl-glyceraldehyde and V K for l-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as d-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V K for 3-APADPH, and V K for d-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.

AB - The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2′-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2′-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V K for NADPH, the V K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2′-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V K for dl-glyceraldehyde and V K for l-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as d-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V K for 3-APADPH, and V K for d-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.

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