Regulation and quaternary structural changes in rabbit muscle phosphofructokinase

Guang Zuan Cai, Lucy L Y Lee, Michael A. Luther, James Lee

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Subunit assembly plays a significant role in the regulation of rabbit muscle phosphofructokinase (PFK), although conformational changes and post-translational modifications have also been implicated to regulate the enzyme activity. In the absence of high-resolution structural information, the three-dimensional arrangements of subunits in the rabbit muscle PFK in its active and inactive states are not known. Hence, a systematic study is initiated, and phosphorylation of PFK subunit is employed as a probe for the structure-function correlation of the enzyme. The self-association of the phosphorylated and dephosphorylated PFK was monitored by sedimentation velocity at pH 7.0 and 23°C. Results show that both the phosphorylated and dephosphorylated forms of PFK exhibit the same mechanism of assembly. The secondary structures of both forms of PFK were monitored by circular dichroism (CD) as a function of protein concentration ranging from 20 to 2000 μ/ml. Results show that there is no detectable difference in the structure under all experimental conditions. The accessibility of tryptophan to solvent was monitored by fluorescence quenching within the same range of protein concentration. Results show that the fluorophores are more accessible to the quencher at higher protein concentrations. Hence, post-translational modification and subunit association do not induce significant structural change in PFK subunit, although the accessibility of tryptophan residues is altered with oligomer formation. Furthermore, sedimentation and CD studies show that the activation of PFK by substrate includes no detectable modification in secondary/tertiary structure but a quaternary structural change, and the localenvironnients of some, if not all, of the tryptophan residues are less accessible to solvent. Hence, the change in sedunentation behavior between the active and inactive tetrameric PFK is due to a rearrangement of subunit-subunit interactions. In order to correlate the physical properties of PFK to the regulatory behavior of enzyme activity, the steady-state kinetics were investigated under the same experimental conditions. In conditions where enhancement of self-association is observed, the kinetic behavior reflects activation of the enzyme. Hence, this correlation between subunit assembly and the regulation of enzyme activity in PFK must reflect an intrinsic property of the muscle enzyme.

Original languageEnglish (US)
Pages (from-to)97-106
Number of pages10
JournalBiophysical Chemistry
Volume37
Issue number1-3
DOIs
StatePublished - Aug 31 1990
Externally publishedYes

Fingerprint

Phosphofructokinases
enzyme activity
tryptophan
rabbits
muscles
Muscle
enzymes
assembly
Rabbits
proteins
Muscles
dichroism
activation
phosphorylation
kinetics
oligomers
Enzyme activity
Tryptophan
physical properties
quenching

Keywords

  • (Rabbit muscle)
  • Enzyme regulation
  • Phosphofructokinase
  • Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Physical and Theoretical Chemistry

Cite this

Regulation and quaternary structural changes in rabbit muscle phosphofructokinase. / Cai, Guang Zuan; Lee, Lucy L Y; Luther, Michael A.; Lee, James.

In: Biophysical Chemistry, Vol. 37, No. 1-3, 31.08.1990, p. 97-106.

Research output: Contribution to journalArticle

Cai, Guang Zuan ; Lee, Lucy L Y ; Luther, Michael A. ; Lee, James. / Regulation and quaternary structural changes in rabbit muscle phosphofructokinase. In: Biophysical Chemistry. 1990 ; Vol. 37, No. 1-3. pp. 97-106.
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AB - Subunit assembly plays a significant role in the regulation of rabbit muscle phosphofructokinase (PFK), although conformational changes and post-translational modifications have also been implicated to regulate the enzyme activity. In the absence of high-resolution structural information, the three-dimensional arrangements of subunits in the rabbit muscle PFK in its active and inactive states are not known. Hence, a systematic study is initiated, and phosphorylation of PFK subunit is employed as a probe for the structure-function correlation of the enzyme. The self-association of the phosphorylated and dephosphorylated PFK was monitored by sedimentation velocity at pH 7.0 and 23°C. Results show that both the phosphorylated and dephosphorylated forms of PFK exhibit the same mechanism of assembly. The secondary structures of both forms of PFK were monitored by circular dichroism (CD) as a function of protein concentration ranging from 20 to 2000 μ/ml. Results show that there is no detectable difference in the structure under all experimental conditions. The accessibility of tryptophan to solvent was monitored by fluorescence quenching within the same range of protein concentration. Results show that the fluorophores are more accessible to the quencher at higher protein concentrations. Hence, post-translational modification and subunit association do not induce significant structural change in PFK subunit, although the accessibility of tryptophan residues is altered with oligomer formation. Furthermore, sedimentation and CD studies show that the activation of PFK by substrate includes no detectable modification in secondary/tertiary structure but a quaternary structural change, and the localenvironnients of some, if not all, of the tryptophan residues are less accessible to solvent. Hence, the change in sedunentation behavior between the active and inactive tetrameric PFK is due to a rearrangement of subunit-subunit interactions. In order to correlate the physical properties of PFK to the regulatory behavior of enzyme activity, the steady-state kinetics were investigated under the same experimental conditions. In conditions where enhancement of self-association is observed, the kinetic behavior reflects activation of the enzyme. Hence, this correlation between subunit assembly and the regulation of enzyme activity in PFK must reflect an intrinsic property of the muscle enzyme.

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