The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase: Kinetic and structural effects of mutations of Phe-50

R. M. Czerwinski, Thomas K Harris, M. A. Massiah, A. S. Mildvan, C. P. Whitman

Research output: Contribution to journalArticle

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Abstract

The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of β,γ-unsaturated enones to their α,β-isomers because of its unusually low pKa of 6.4 ± 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pKa is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358-12366]. Hence, it may result solely from a low local dielectric constant of 14.7 ± 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 Å from Pro-1 and is one of 12 apolar residues within 9 Å of Pro-1. Replacing Phe-50 with Tyr does not significantly alter kcat or Km and results in a pKa of 6.0 ± 0.1 for Pro-1 as determined by 15N NMR spectroscopy, comparable to that observed for wild type. 1H-15N HSQC and 3D 1H-15N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pKa of Tyr-50 is increased by two units from that of a model compound N-acetyltyrosine amide to 12.2 ± 0.3, as determined by UV and 1H NMR titrations, yielding a local dielectric constant of 13.4 ± 1.7, in agreement with the value of 13.7 ± 0.3 determined from the decreased pKa of Pro-1 in this mutant. In the F50A mutant, the pKa of Pro-1 is 7.3 ± 0.1 by 15N NMR titration, comparable to the pKa of 7.6 ± 0.2 found in the pH vs kcat/Km rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 ± 2.6. A loss of structure of the β-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. 1H-15N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone 15N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 °C and their disappearance at 43 °C due to rapid exchange with solvent. These observations confirm that the active site of the F5OA mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pKa of Pro-1. In addition, the F50A mutation decreased kcat 167-fold and increased Km 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pKa of Pro-1. The F50I and F50V mutations destabilize the protein and decrease kcat by factors of 58 and 1.6, and increase Km by 3.3- and 3.8-fold, respectively.

Original languageEnglish
Pages (from-to)1984-1995
Number of pages12
JournalBiochemistry
Volume40
Issue number7
DOIs
StatePublished - Feb 20 2001

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Catalytic Domain
Permittivity
Mutation
Kinetics
Enzymes
Titration
Proline
Amides
Nuclear magnetic resonance
Biochemistry
Chemical shift
Isomerization
Static Electricity
Catalysis
Isomers
Nuclear magnetic resonance spectroscopy
Conformations
Electrostatics
Magnetic Resonance Spectroscopy
Catalysts

ASJC Scopus subject areas

  • Biochemistry

Cite this

The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase : Kinetic and structural effects of mutations of Phe-50. / Czerwinski, R. M.; Harris, Thomas K; Massiah, M. A.; Mildvan, A. S.; Whitman, C. P.

In: Biochemistry, Vol. 40, No. 7, 20.02.2001, p. 1984-1995.

Research output: Contribution to journalArticle

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title = "The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase: Kinetic and structural effects of mutations of Phe-50",
abstract = "The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of β,γ-unsaturated enones to their α,β-isomers because of its unusually low pKa of 6.4 ± 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pKa is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358-12366]. Hence, it may result solely from a low local dielectric constant of 14.7 ± 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 {\AA} from Pro-1 and is one of 12 apolar residues within 9 {\AA} of Pro-1. Replacing Phe-50 with Tyr does not significantly alter kcat or Km and results in a pKa of 6.0 ± 0.1 for Pro-1 as determined by 15N NMR spectroscopy, comparable to that observed for wild type. 1H-15N HSQC and 3D 1H-15N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pKa of Tyr-50 is increased by two units from that of a model compound N-acetyltyrosine amide to 12.2 ± 0.3, as determined by UV and 1H NMR titrations, yielding a local dielectric constant of 13.4 ± 1.7, in agreement with the value of 13.7 ± 0.3 determined from the decreased pKa of Pro-1 in this mutant. In the F50A mutant, the pKa of Pro-1 is 7.3 ± 0.1 by 15N NMR titration, comparable to the pKa of 7.6 ± 0.2 found in the pH vs kcat/Km rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 ± 2.6. A loss of structure of the β-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. 1H-15N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone 15N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 °C and their disappearance at 43 °C due to rapid exchange with solvent. These observations confirm that the active site of the F5OA mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pKa of Pro-1. In addition, the F50A mutation decreased kcat 167-fold and increased Km 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pKa of Pro-1. The F50I and F50V mutations destabilize the protein and decrease kcat by factors of 58 and 1.6, and increase Km by 3.3- and 3.8-fold, respectively.",
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T1 - The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase

T2 - Kinetic and structural effects of mutations of Phe-50

AU - Czerwinski, R. M.

AU - Harris, Thomas K

AU - Massiah, M. A.

AU - Mildvan, A. S.

AU - Whitman, C. P.

PY - 2001/2/20

Y1 - 2001/2/20

N2 - The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of β,γ-unsaturated enones to their α,β-isomers because of its unusually low pKa of 6.4 ± 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pKa is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358-12366]. Hence, it may result solely from a low local dielectric constant of 14.7 ± 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 Å from Pro-1 and is one of 12 apolar residues within 9 Å of Pro-1. Replacing Phe-50 with Tyr does not significantly alter kcat or Km and results in a pKa of 6.0 ± 0.1 for Pro-1 as determined by 15N NMR spectroscopy, comparable to that observed for wild type. 1H-15N HSQC and 3D 1H-15N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pKa of Tyr-50 is increased by two units from that of a model compound N-acetyltyrosine amide to 12.2 ± 0.3, as determined by UV and 1H NMR titrations, yielding a local dielectric constant of 13.4 ± 1.7, in agreement with the value of 13.7 ± 0.3 determined from the decreased pKa of Pro-1 in this mutant. In the F50A mutant, the pKa of Pro-1 is 7.3 ± 0.1 by 15N NMR titration, comparable to the pKa of 7.6 ± 0.2 found in the pH vs kcat/Km rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 ± 2.6. A loss of structure of the β-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. 1H-15N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone 15N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 °C and their disappearance at 43 °C due to rapid exchange with solvent. These observations confirm that the active site of the F5OA mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pKa of Pro-1. In addition, the F50A mutation decreased kcat 167-fold and increased Km 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pKa of Pro-1. The F50I and F50V mutations destabilize the protein and decrease kcat by factors of 58 and 1.6, and increase Km by 3.3- and 3.8-fold, respectively.

AB - The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of β,γ-unsaturated enones to their α,β-isomers because of its unusually low pKa of 6.4 ± 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pKa is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358-12366]. Hence, it may result solely from a low local dielectric constant of 14.7 ± 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 Å from Pro-1 and is one of 12 apolar residues within 9 Å of Pro-1. Replacing Phe-50 with Tyr does not significantly alter kcat or Km and results in a pKa of 6.0 ± 0.1 for Pro-1 as determined by 15N NMR spectroscopy, comparable to that observed for wild type. 1H-15N HSQC and 3D 1H-15N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pKa of Tyr-50 is increased by two units from that of a model compound N-acetyltyrosine amide to 12.2 ± 0.3, as determined by UV and 1H NMR titrations, yielding a local dielectric constant of 13.4 ± 1.7, in agreement with the value of 13.7 ± 0.3 determined from the decreased pKa of Pro-1 in this mutant. In the F50A mutant, the pKa of Pro-1 is 7.3 ± 0.1 by 15N NMR titration, comparable to the pKa of 7.6 ± 0.2 found in the pH vs kcat/Km rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 ± 2.6. A loss of structure of the β-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. 1H-15N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone 15N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 °C and their disappearance at 43 °C due to rapid exchange with solvent. These observations confirm that the active site of the F5OA mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pKa of Pro-1. In addition, the F50A mutation decreased kcat 167-fold and increased Km 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pKa of Pro-1. The F50I and F50V mutations destabilize the protein and decrease kcat by factors of 58 and 1.6, and increase Km by 3.3- and 3.8-fold, respectively.

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