pH Dependency of the Zinc and Cobalt Carboxypeptidase Catalyzed Enolization of (R)-2-Benzyl-3-(p-methoxybenzoyl)propionic Acid

Thomas Spratt, Takuji Sugimoto, E. T. Kaiser

Research output: Contribution to journalArticle

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Abstract

The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyljpropionic acid ((-)-1) has been determined. The study of this relatively simple reaction allows us to examine the catalytic properties and ionization behavior of the enzyme-bound active-site bases and acids without the complications that would be encountered for peptides and esters where the formation and breakdown of a multiplicity of intermediates along the reaction pathway must be considered. The pKa values of 6.03 ± 0.35 and 6.04 ± 0.31 measured from the pH dependency of kcat for the Zn(II) and Co(II) CPA catalyzed enolization of (-)-1, respectively, correspond to the pka for the ionization of the γ-carboxyl of Glu-270. The binding of (-)-1 to CPA was investigated by examining the inhibition by this compound of the enzyme-catalyzed hydrolysis of O-(trans-p-chlorocinnamoyl)-L-β-phenyllactate. The KI-pH dependencies for the inhibitory activity of (-)-1 show that in alkaline solution one ionization of an enzyme-bound group occuss with pKa = 7.56 ±0.15 for the Zn(II) enzyme and pKa = 8.29 ± 0.32 for Co(II) CPA while the other occuss above pH 9. The pKa values in the vicinity of 8 represent the ionization of the phenolic hydroxyl of Tyr-248, in good agreement with earlier assignments of this pK. Finally, a reasonable interpretation of the pH dependency on a group with pKa <9 is that it corresponds to the ionization of the active-site metal ion bound water.

Original languageEnglish (US)
Pages (from-to)3679-3683
Number of pages5
JournalJournal of the American Chemical Society
Volume105
Issue number11
DOIs
StatePublished - Jan 1 1983

Fingerprint

Carboxypeptidases
Propionic acid
Cobalt
Ionization
Zinc
Enzymes
Catalytic Domain
Enzyme inhibition
Acids
Hydroxyl Radical
Peptides
Metal ions
Hydrolysis
Esters
Metals
propionic acid
Ions
Kinetics
Water
Substrates

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

@article{707e6053cb34467f98dd058ba91d933a,
title = "pH Dependency of the Zinc and Cobalt Carboxypeptidase Catalyzed Enolization of (R)-2-Benzyl-3-(p-methoxybenzoyl)propionic Acid",
abstract = "The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyljpropionic acid ((-)-1) has been determined. The study of this relatively simple reaction allows us to examine the catalytic properties and ionization behavior of the enzyme-bound active-site bases and acids without the complications that would be encountered for peptides and esters where the formation and breakdown of a multiplicity of intermediates along the reaction pathway must be considered. The pKa values of 6.03 ± 0.35 and 6.04 ± 0.31 measured from the pH dependency of kcat for the Zn(II) and Co(II) CPA catalyzed enolization of (-)-1, respectively, correspond to the pka for the ionization of the γ-carboxyl of Glu-270. The binding of (-)-1 to CPA was investigated by examining the inhibition by this compound of the enzyme-catalyzed hydrolysis of O-(trans-p-chlorocinnamoyl)-L-β-phenyllactate. The KI-pH dependencies for the inhibitory activity of (-)-1 show that in alkaline solution one ionization of an enzyme-bound group occuss with pKa = 7.56 ±0.15 for the Zn(II) enzyme and pKa = 8.29 ± 0.32 for Co(II) CPA while the other occuss above pH 9. The pKa values in the vicinity of 8 represent the ionization of the phenolic hydroxyl of Tyr-248, in good agreement with earlier assignments of this pK. Finally, a reasonable interpretation of the pH dependency on a group with pKa <9 is that it corresponds to the ionization of the active-site metal ion bound water.",
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pH Dependency of the Zinc and Cobalt Carboxypeptidase Catalyzed Enolization of (R)-2-Benzyl-3-(p-methoxybenzoyl)propionic Acid. / Spratt, Thomas; Sugimoto, Takuji; Kaiser, E. T.

In: Journal of the American Chemical Society, Vol. 105, No. 11, 01.01.1983, p. 3679-3683.

Research output: Contribution to journalArticle

TY - JOUR

T1 - pH Dependency of the Zinc and Cobalt Carboxypeptidase Catalyzed Enolization of (R)-2-Benzyl-3-(p-methoxybenzoyl)propionic Acid

AU - Spratt, Thomas

AU - Sugimoto, Takuji

AU - Kaiser, E. T.

PY - 1983/1/1

Y1 - 1983/1/1

N2 - The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyljpropionic acid ((-)-1) has been determined. The study of this relatively simple reaction allows us to examine the catalytic properties and ionization behavior of the enzyme-bound active-site bases and acids without the complications that would be encountered for peptides and esters where the formation and breakdown of a multiplicity of intermediates along the reaction pathway must be considered. The pKa values of 6.03 ± 0.35 and 6.04 ± 0.31 measured from the pH dependency of kcat for the Zn(II) and Co(II) CPA catalyzed enolization of (-)-1, respectively, correspond to the pka for the ionization of the γ-carboxyl of Glu-270. The binding of (-)-1 to CPA was investigated by examining the inhibition by this compound of the enzyme-catalyzed hydrolysis of O-(trans-p-chlorocinnamoyl)-L-β-phenyllactate. The KI-pH dependencies for the inhibitory activity of (-)-1 show that in alkaline solution one ionization of an enzyme-bound group occuss with pKa = 7.56 ±0.15 for the Zn(II) enzyme and pKa = 8.29 ± 0.32 for Co(II) CPA while the other occuss above pH 9. The pKa values in the vicinity of 8 represent the ionization of the phenolic hydroxyl of Tyr-248, in good agreement with earlier assignments of this pK. Finally, a reasonable interpretation of the pH dependency on a group with pKa <9 is that it corresponds to the ionization of the active-site metal ion bound water.

AB - The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyljpropionic acid ((-)-1) has been determined. The study of this relatively simple reaction allows us to examine the catalytic properties and ionization behavior of the enzyme-bound active-site bases and acids without the complications that would be encountered for peptides and esters where the formation and breakdown of a multiplicity of intermediates along the reaction pathway must be considered. The pKa values of 6.03 ± 0.35 and 6.04 ± 0.31 measured from the pH dependency of kcat for the Zn(II) and Co(II) CPA catalyzed enolization of (-)-1, respectively, correspond to the pka for the ionization of the γ-carboxyl of Glu-270. The binding of (-)-1 to CPA was investigated by examining the inhibition by this compound of the enzyme-catalyzed hydrolysis of O-(trans-p-chlorocinnamoyl)-L-β-phenyllactate. The KI-pH dependencies for the inhibitory activity of (-)-1 show that in alkaline solution one ionization of an enzyme-bound group occuss with pKa = 7.56 ±0.15 for the Zn(II) enzyme and pKa = 8.29 ± 0.32 for Co(II) CPA while the other occuss above pH 9. The pKa values in the vicinity of 8 represent the ionization of the phenolic hydroxyl of Tyr-248, in good agreement with earlier assignments of this pK. Finally, a reasonable interpretation of the pH dependency on a group with pKa <9 is that it corresponds to the ionization of the active-site metal ion bound water.

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