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Biochemistry. 1995 Jul 4;34(26):8309-19.

Dynamics of the flexible loop of triosephosphate isomerase: the loop motion is not ligand gated.

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  • 1Department of Chemistry, Columbia University, New York, New York 10027, USA.

Abstract

Using solid-state deuterium NMR, we have measured the motion of the flexible loop of triosephosphate isomerase (TIM) with and without substrate and transition-state analogs. The measurements were carried out on a catalytically competent mutant of TIM W90Y W157F containing a single tryptophan (W168) in the flexible loop; W168 is the only strictly conserved tryptophan in the currently available TIM sequences. The solid-state NMR samples were prepared by precipitation using polyethylene glycol, and kinetic analysis of the PEG-precipitated TIM gave values for kcat, Km, and KI similar to those measured in solution for the substrate and substrate and transition-state analogs. Deuterium NMR spectra of samples prepared with tryptophan labeled at the indole positions with and without any substrate or analogs indicate that the loop jumps between two conformations at a rate of 3 x 10(4) s-1 (from the predominant to the less populated form) with a population ratio of 10:1. Surprisingly, spectra of TIM ligated with a substrate analog, glycerol 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 ratio for the two conformers. This observation indicates that loop closure is not ligand gated but is a natural motion of the protein. Furthermore, the measured rate is approximately matched to the turnover time. We did not observe a signal for TIM labeled with alpha-deuteriotryptophan, although it was prepared in a fashion analogous to the ring-labeled sample and had a specific activity and protein concentration comparable to the latter. For this deuterium concentration, we would expect to observe the NMR signal unless the deuterium relaxation were very slow. The hypothesis that the spin-lattice relaxation of the alpha-deuteron is very slow would be consistent with the observed dynamics of the ring-deuterated TIM.

PMID:
7599123
[PubMed - indexed for MEDLINE]
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