Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1990 Aug 31;37(1-3):97-106.
doi: 10.1016/0301-4622(90)88011-g.

Regulation and quaternary structural changes in rabbit muscle phosphofructokinase

Affiliations

Regulation and quaternary structural changes in rabbit muscle phosphofructokinase

G Z Cai et al. Biophys Chem. .

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 degrees 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 micrograms/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 local environments of some, if not all, of the tryptophan residues are less accessible to solvent. Hence, the change in sedimentation 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.

PubMed Disclaimer

Publication types

LinkOut - more resources