1i45
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(New page: 200px<br /><applet load="1i45" size="450" color="white" frame="true" align="right" spinBox="true" caption="1i45, resolution 1.80Å" /> '''YEAST TRIOSEPHOSPHAT...)
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Revision as of 14:55, 20 November 2007
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YEAST TRIOSEPHOSPHATE ISOMERASE (MUTANT)
Overview
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 19F and 31P 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 degrees C. In contrast, a single NMR peak, representing a rapid average of the two species was observed at 30 degrees, C. We conclude that the rate of loop motion is less than 1400 s(-1) at 0, degrees C and more than 1400 s(-1) at 30 degrees C. Ligand release was, studied under similar sample conditions, using 31P NMR of the phosphate, group of the substrate analogue. The rate of ligand release is less than, 1000 s(-1) at 0 degrees C and more than 1000 s(-1) at 30 degrees C., Therefore, loop motion and product release are probably concerted and, likely to represent a rate limiting step for chemistry.
About this Structure
1I45 is a Single protein structure of sequence from Saccharomyces cerevisiae. Active as Triose-phosphate isomerase, with EC number 5.3.1.1 Full crystallographic information is available from OCA.
Reference
Solution-state NMR investigations of triosephosphate isomerase active site loop motion: ligand release in relation to active site loop dynamics., Rozovsky S, Jogl G, Tong L, McDermott AE, J Mol Biol. 2001 Jun 29;310(1):271-80. PMID:11419952
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