Monitoring Active Site Alterations upon Mutation of Yeast Pyruvate Kinase Using 205Tl+ NMR

Abstract

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 Mn 2+ 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/T1 value of 205Tl+ 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/pT 1p 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 Mn 2+-205Tl+ interaction. The Tl +-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to 1/T 1M 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.