Phenylalanine Hydroxylase from Chromobacterium violaceum Is a Copper-Containing Monooxygenase. Kinetics of the Reductive Activation of the Enzyme

Stephen O. Pember, Joseph J. Villafranca, Stephen Benkovic

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.

Original languageEnglish (US)
Pages (from-to)6611-6619
Number of pages9
JournalBiochemistry
Volume25
Issue number21
DOIs
StatePublished - Jan 1 1986

Fingerprint

Chromobacterium
Phenylalanine Hydroxylase
Enzyme Activation
Mixed Function Oxygenases
Copper
Chemical activation
Kinetics
Enzymes
Dithiothreitol
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Pterins
Reducing Agents
Hydrates
Sulfhydryl Compounds
Spectrum Analysis

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Pember, Stephen O. ; Villafranca, Joseph J. ; Benkovic, Stephen. / Phenylalanine Hydroxylase from Chromobacterium violaceum Is a Copper-Containing Monooxygenase. Kinetics of the Reductive Activation of the Enzyme. In: Biochemistry. 1986 ; Vol. 25, No. 21. pp. 6611-6619.
@article{816dd43558b242cb9f6808b4e53489a7,
title = "Phenylalanine Hydroxylase from Chromobacterium violaceum Is a Copper-Containing Monooxygenase. Kinetics of the Reductive Activation of the Enzyme",
abstract = "Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.",
author = "Pember, {Stephen O.} and Villafranca, {Joseph J.} and Stephen Benkovic",
year = "1986",
month = "1",
day = "1",
doi = "10.1021/bi00369a042",
language = "English (US)",
volume = "25",
pages = "6611--6619",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "21",

}

Phenylalanine Hydroxylase from Chromobacterium violaceum Is a Copper-Containing Monooxygenase. Kinetics of the Reductive Activation of the Enzyme. / Pember, Stephen O.; Villafranca, Joseph J.; Benkovic, Stephen.

In: Biochemistry, Vol. 25, No. 21, 01.01.1986, p. 6611-6619.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phenylalanine Hydroxylase from Chromobacterium violaceum Is a Copper-Containing Monooxygenase. Kinetics of the Reductive Activation of the Enzyme

AU - Pember, Stephen O.

AU - Villafranca, Joseph J.

AU - Benkovic, Stephen

PY - 1986/1/1

Y1 - 1986/1/1

N2 - Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.

AB - Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.

UR - http://www.scopus.com/inward/record.url?scp=0023038339&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023038339&partnerID=8YFLogxK

U2 - 10.1021/bi00369a042

DO - 10.1021/bi00369a042

M3 - Article

C2 - 3024714

AN - SCOPUS:0023038339

VL - 25

SP - 6611

EP - 6619

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 21

ER -