Dissociative mechanism of thermal denaturation of rabbit skeletal muscle glycogen phosphorylase b.
The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53 degrees C occurs after cooling of the enzyme solution to 30 degrees C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.
Kurganov
B I
BI
A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospekt 33, Moscow 117071, Russian Federation.
Kornilaev
B A
BA
Chebotareva
N A
NA
Malikov
V P
VP
Orlov
V N
VN
Lyubarev
A E
AE
Livanova
N B
NB
D000818:Animals; D002152:Calorimetry, Differential Scanning; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D006003:Glycogen / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008024:Ligands; D018482:Muscle, Skeletal / Q000201:enzymology*; D010763:Phosphorylase b / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D013552:Swine; D014461:Ultracentrifugation
D004793:Enzyme Reactivators; D008024:Ligands; D011732:Pyridoxal Phosphate; D006003:Glycogen; D010763:Phosphorylase b