Effects of metabolites on the structural dynamics of rabbit muscle pyruvate kinase.
The activity of rabbit muscle pyruvate kinase (PK) is regulated by metabolites. Besides requiring the presence of its substrates, PEP and ADP, the enzyme requires Mg(2+) and K(+) for activity. PK is allosterically inhibited by Phe for activity. The presence of PEP or Phe has opposing effects on the hydrodynamic properties of the enzyme without an apparent change in secondary structure. In this study, the structural perturbation induced by ligand binding was investigated by Fourier transform infrared (FT-IR) spectroscopy. Furthermore, the structural dynamics of PK was probed by H/D exchange monitored by FT-IR. Substrates and activating metal ions induce PK to assume a more dynamic structure while Phe exerts an opposite effect. In all cases there is no significant interconversion of secondary structures. PEP is the most efficient ligand in inducing a change in the microenvironments of both helices and sheets so much so that they can be detected spectroscopically as separate bands. These results provide the first evidence for a differential effect of ligand binding on the dynamics of structural elements in PK. Furthermore, the data support the model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the observed change in hydrodynamic properties represent the two extreme end states.
Yu
Shaoning
S
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-1055, USA.
Lee
Lucy L-Y
LL
Lee
J Ching
JC
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D002942:Circular Dichroism; D007700:Kinetics; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology*; D010649:Phenylalanine / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D017550:Spectroscopy, Fourier Transform Infrared
D010649:Phenylalanine; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase