title: Conformational changes in yeast pyruvate kinase studied by 205Tl+ NMR.
abstract: The interaction of the monovalent cation with yeast pyruvate kinase (yPK) has been investigated by 205Tl+ NMR. TlNO3 activates yPK to 80-90% activity compared to KCl with an apparent Ka of 1.00 +/- 0.03 mM in the presence of 4 mM Mn(NO3)2 as the activating divalent cation. At higher concentrations of Tl+, enzyme inhibition is observed with an apparent KI of 180 +/- 10 mM. The extent of inhibition is dependent on the nature and concentration of the divalent cation. The effect of Mn2+ on the 1/T1 and 1/T2 values of 205Tl+ in the presence of yPK was determined at 173.02 MHz (300 MHz, 1H) and 346.03 MHz (600 MHz, 1H). The temperature dependence of the relaxation rates indicates that fast exchange conditions prevail for 205Tl+ longitudinal relaxation rates. The correlation time, tauc, for the Mn2+-205Tl+ interaction was estimated by a frequency dependence of 1/T1m for several enzyme complexes, and an average value of tauc was determined to be 0.91 ns. The distance between Tl+ and Mn2+ at the active site of yPK was calculated from the paramagnetic contribution of Mn2+ to the longitudinal (1/T1m) relaxation rates of Tl+ bound to yPK. For the apo yPK complex, the Tl+ to Mn2+ distance is 6.7 +/- 0.2 A. Upon addition of phosphoenolpyruvate (PEP) to form the yPK-Tl-Mn-PEP complex, the inter-cation distance decreases to 6.1 +/- 0.3 A. The addition of the allosteric activator fructose 1,6-bisphosphate (FBP) to form the yPK-Tl+-Mn2+-PEP-FBP complex gives an intermetal distance of 6.2 +/- 0.2 A. In the yPK-Tl-Mn-FBP complex, a Tl+-Mn2+ distance of 6.0 +/- 0.1 A is observed, indicating that FBP causes a conformational change at the active site in the absence of PEP. Analogous 205Tl NMR experiments with competitive inhibitors of PEP (oxalate, BrPEP) indicate that these ligands do not induce the same conformational changes as do the physiological substrates and activators. Similar experiments with the nonallosteric rabbit muscle PK were also performed and analyzed.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Loria
authors.forename: T
authors.initials: T
authors.lastname: Nowak
mesh_terms: D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D018482:Muscle, Skeletal / Q000201:enzymology; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013793:Thallium
chemical_list: D008345:Manganese; D013793:Thallium; D011770:Pyruvate Kinase