Solution-state NMR investigations of triosephosphate isomerase active site loop motion: Ligand release in relation to active site loop dynamics

Product release is partially rate determining in the isomerization reaction catalyzed by Triosephosphate Isomerase, the conversion of dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate, probably because an active-site loop movement is necessary to free the product from confinement in the active-site. The timescale of the catalytic loop motion and of ligand release were studied using F-19 and P-31 solution-state NMR. A 5 ' -fluorotryptophan was incorporated in the loop N-terminal hinge as a reporter of loop motion timescale. Crystallographic studies confirmed that the structure of the fluorinated enzyme is indistinguishable from the wild-type; the fluorine accepts a hydrogen bond from water and not from a protein residue, with minimal perturbation to the flexible loop stability. Two distinct loop conformations were observed by 19F NMR. Both for unligated (empty) and ligated enzyme samples a single species was detected, but the chemical shifts of these two distinct species differed by 1.2 ppm. For samples in the presence of subsaturating amounts of a substrate analogue, glycerol 3-phosphate, both NMR peaks were present, with broadened lineshapes at 0 degreesC. Ln contrast, a single NMR peak representing a rapid average of the two species was observed at 30 degreesC. We conclude that the rate of loop motion is less than 1400 s(-1) at 0 degreesC and more than 1400 s(-1) at 30 degreesC. Ligand release was studied under similar sample conditions, using (31)p NMR Of the phosphate group of the substrate analogue. The rate of ligand release is less than 1000 s(-1) at 0 degreesC and more than 1000 s(-1) at 30 degreesC. Therefore, loop motion and product release are probably concerted and likely to represent a rate limiting step for chemistry. (C) 2001 Academic Press.