Domain interaction in rabbit muscle pyruvate kinase. I. Effects of ligands on protein denaturation induced by guanidine hydrochloride.
The structural stability of rabbit muscle pyruvate kinase was examined. The unfolding of pyruvate kinase was induced by guanidine hydrochloride, and the process was monitored by spectroscopic techniques (fluorescence and UV absorption) and hydrodynamic measurements (sedimentation velocity, sedimentation equilibrium, densimetry, and viscometry). The spectroscopic techniques revealed that the unfolding of pyruvate kinase induced by guanidine hydrochloride is not a simple cooperative process. This suggests that different regions of pyruvate kinase are unfolding with different efficiencies in response to the denaturant. These regions are most likely related to the domain structures observed by x-ray crystallography. In the presence of L-phenylalanine, the allosteric inhibitor, the denaturation process became more cooperative, and the enzyme dissociated and unfolded at a higher denaturant concentration. The binding of phenylalanine also induced a structural change in the enzyme, rendering it more susceptible to tryptic digestion. One of the peptides, the production rate of which was increased, was isolated and sequenced. Its N terminus is located at the interface between two domains, one of which contains the active site. This evidence indicates structural changes, probably involving domain-domain interaction, for pyruvate kinase in response to phenylalanine binding.
Consler
T G
TG
E. A. Doisy Department of Biochemistry, St. Louis University School of Medicine, Missouri 63104.
Lee
J C
JC
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002415:Cats; D002645:Chickens; D019791:Guanidine; D006146:Guanidines; D007527:Isoenzymes / Q000032:analysis; D008024:Ligands; D009132:Muscles / Q000201:enzymology*; D010449:Peptide Mapping; D011489:Protein Denaturation; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; D007527:Isoenzymes; D008024:Ligands; D011770:Pyruvate Kinase; D019791:Guanidine