Changes in small-angle X-ray scattering parameters observed upon binding of ligand to rabbit muscle pyruvate kinase are not correlated with allosteric transitions.
Protein fluorescence and small-angle X-ray scattering (SAXS) have been used to monitor effector affinity and conformational changes previously associated with allosteric regulation in rabbit muscle pyruvate kinase (M(1)-PYK). In the absence of substrate [phosphoenolpyruvate (PEP)], SAXS-monitored conformational changes in M(1)-PYK elicited by the binding of phenylalanine (an allosteric inhibitor that reduces the affinity of M(1)-PYK for PEP) are similar to those observed upon binding of alanine or 2-aminobutyric acid. Under our assay conditions, these small amino acids bind to the protein but elicit a minimal change in the affinity of the protein for PEP. Therefore, if changes in scattering signatures represent cleft closure via domain rotation as previously interpreted, we can conclude that these motions are not sufficient to elicit allosteric inhibition. Additionally, although PEP has similar affinities for the free enzyme and the M(1)-PYK-small amino acid complexes (i.e., the small amino acids have minimal allosteric effects), PEP binding elicits different changes in the SAXS signature of the free enzyme versus the M(1)-PYK-small amino acid complexes.
Fenton
Aron W
AW
Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, MS 3030, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA. afenton@kumc.edu
Williams
Rachel
R
Trewhella
Jill
J
D000494:Allosteric Regulation; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D005453:Fluorescence; D008024:Ligands; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology*; D010649:Phenylalanine / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits / Q000378:metabolism*; D053838:Scattering, Small Angle; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D000596:Amino Acids; D008024:Ligands; D010649:Phenylalanine; D011770:Pyruvate Kinase