DYNAMICS OF THE FLEXIBLE LOOP OF TRIOSEPHOSPHATE ISOMERASE - THE LOOPMOTION IS NOT LIGAND-GATED

Using solid-state deuterium NMR, we have measured the motion of the fl exible loop of triosephosphate isomerase (TIM) with and without substr ate and transition-state analogs, The measurements were carried out on a catalytically competent mutant of TIM W90Y W157F containing a singl e tryptophan (W168) in the flexible loop; W168 is the only strictly co nserved tryptophan in the currently available TIM sequences. The solid -state NMR samples were prepared by precipitation using polyethylene g lycol, and kinetic analysis of the PEG-precipitated TIM gave values fo r k(cat), K-m, and K-I similar to those measured in solution for the s ubstrate and substrate and transition-state analogs, Deuterium NMR spe ctra of samples prepared with tryptophan labeled at the indole positio ns with and without any substrate or analogs indicate that the loop ju mps between two conformations at a rate of 3 x 10(4) s(-1) (from the p redominant to the less populated form) with a population ratio of 10:1 . Surprisingly, spectra of TIM ligated with a substrate analog, glycer ol 3-phosphate (G3P), or with a tight-binding transition-state analog, phosphoglycolate (PGA), show that the loop moves with a rate similar to the rate in the empty enzyme and also has a similar population rati o for the two conformers. This observation indicates that loop closure is not ligand gated but is a natural motion of the protein. Furthermo re, the measured rate is approximately matched to the turnover time. W e did not observe a signal for TIM labeled with alpha-deuteriotryptoph an, although it was prepared in a fashion analogous to the ring-labele d sample and had a specific activity and protein concentration compara ble to the latter. For this deuterium concentration, we would expect t o observe the NMR signal unless the deuterium relaxation were very slo w. The hypothesis that the spin-lattice relaxation of the alpha-deuter on is very slow would be consistent with the observed dynamics of the ring-deuterated TIM.