Modification of the regulatory properties of pyruvate kinase of Neurospora by growth at elevated temperatures.
Pyruvate kinase (EC 2.7.1.40) was isolated from Neurospora crassa mycelium grown at 28 degrees C (PK-28) and at 42 degrees C (PK-42). The regulatory properties, particularly the response towards the allosteric effector fructose 1,6-diphosphate (FDP), was different in the two enzymes. PK-28 showed an activation by FDP but PK-42, under comparable conditions, appeared to be activated by low concentrations of FDP and inhibited by higher ones. For PK-28, complex formation with FDP results in a lowering of the isoelectric point from 6.40 to 5.50, representing the pI of the unliganded enzyme and that of the complex, respectively. In contrast to this, PK-42 exhibits a weak binding to FDP as suggested by a lack of decrease in the isoelectric point on treatment with comparable concentrations of FDP. Studies with quenching of aromatic residue fluorescence of PK-28 and PK-42, following binding of FDP, indicate that although this ligand binds to both types of enzymes the affinity for the two is vastly different. Dissociation constants of 9.3 muM and 0.1 mM were calculated for the binding of FDP to PK-28 and PK-42, respectively. It is concluded that growth at elevated temperatures induced a conformational change in the pyruvate kinase leading to partial desensitization of the allosteric site. The nature of the factor(s) responsible for this change is not understood at present.
D000494:Allosteric Regulation; D000495:Allosteric Site; D004789:Enzyme Activation / Q000187:drug effects; D005636:Fructosephosphates / Q000494:pharmacology; D006598:Hexosediphosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D005636:Fructosephosphates; D006598:Hexosediphosphates; D011770:Pyruvate Kinase