Monitoring active site alterations upon mutation of yeast pyruvate kinase using 205Tl+ NMR.
The interaction of the monovalent cation with wild type (WT) yeast pyruvate kinase (YPK) and with the T298S, T298C, and T298A mutants was investigated by 205Tl+ NMR to monitor possible structural alterations at the active site by Thr-298 mutation. TlNO3 activates WT YPK with a kcat value similar to that obtained with KCl and an apparent Ka of 0.96 +/- 0.07 mm in the presence of Mn2+ and fructose 1,6-bisphosphate. With the three mutants, Tl+ is a better activator than is K+ based on kcat values. Tl+ activation and inhibition of YPK is affected by mutation of the active site Thr-298. The effect of Mn2+ on the 1/T value of 205Tl+1 in the presence of the WT and mutant YPK complexes was determined at 173 MHz (300 MHz, 1H) and 346 MHz (600 MHz, 1H). For each complex studied, 1/pT2p >> 1/pT1p and 1/pT1p is frequency-dependent suggesting fast exchange conditions. The values of 1/pT1p differ for each mutant. A correlation time of 0.65 +/- 0.35 ns was estimated for the Mn2+-205Tl+ interaction. The Tl+-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to 1/T1M of YPK-bound 205Tl+. The calculated Tl+-Mn2+ distance for the Thr-298 mutants is decreased by about 1 A from 6.0 +/- 0.2 A observed with WT. The results suggest conformational alterations at the active site of YPK where phosphoryl transfer occurs upon mutation of Thr-298. These conformational changes may, in part, explain the alteration in kcat and kcat/Km,PEP observed with the Thr-298 mutants.
Susan-Resiga
Delia
D
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Nowak
Thomas
T
D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009154:Mutation; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry / Q000235:genetics*; D011817:Rabbits; D013794:Thallium Radioisotopes / Q000378:metabolism
D005656:Fungal Proteins; D013794:Thallium Radioisotopes; D011770:Pyruvate Kinase