Human erythrocyte glutathione reductase: pH dependence of kinetic parameters

Kenny K. Wong, John S. Blanchard

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH 2′,3′-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2′,3′-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's s for ATP-ribose and 2′,5′-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2′-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467′-Glu-472′ ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.

Original languageEnglish (US)
Pages (from-to)3586-3590
Number of pages5
JournalBiochemistry
Volume28
Issue number8
StatePublished - 1989

Fingerprint

Glutathione Reductase
NADP
Kinetic parameters
Erythrocytes
Glutathione Disulfide
NAD
Ionization
Enthalpy
Nucleotides
Proton transfer
Ribose
Protonation
Substrates
Adenosine Diphosphate
Adenosine Triphosphate
Phosphates
Ions
Temperature
Enzymes
Protons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human erythrocyte glutathione reductase : pH dependence of kinetic parameters. / Wong, Kenny K.; Blanchard, John S.

In: Biochemistry, Vol. 28, No. 8, 1989, p. 3586-3590.

Research output: Contribution to journalArticle

@article{f65a2c71ea074dd68e7ac1a22c2582d0,
title = "Human erythrocyte glutathione reductase: pH dependence of kinetic parameters",
abstract = "Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH 2′,3′-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2′,3′-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's s for ATP-ribose and 2′,5′-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2′-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467′-Glu-472′ ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.",
author = "Wong, {Kenny K.} and Blanchard, {John S.}",
year = "1989",
language = "English (US)",
volume = "28",
pages = "3586--3590",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - Human erythrocyte glutathione reductase

T2 - pH dependence of kinetic parameters

AU - Wong, Kenny K.

AU - Blanchard, John S.

PY - 1989

Y1 - 1989

N2 - Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH 2′,3′-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2′,3′-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's s for ATP-ribose and 2′,5′-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2′-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467′-Glu-472′ ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.

AB - Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH 2′,3′-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2′,3′-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's s for ATP-ribose and 2′,5′-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2′-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467′-Glu-472′ ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.

UR - http://www.scopus.com/inward/record.url?scp=0024577448&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024577448&partnerID=8YFLogxK

M3 - Article

C2 - 2742856

AN - SCOPUS:0024577448

VL - 28

SP - 3586

EP - 3590

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 8

ER -