Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli.
Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)-3H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the "exchange-conversion" experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.(ABSTRACT TRUNCATED AT 250 WORDS)
Nickbarg
E B
EB
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Knowles
J R
JR
D000818:Animals; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002645:Chickens; D003001:Cloning, Molecular; D003903:Deuterium; D004099:Dihydroxyacetone Phosphate; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*; D014316:Tritium
D014316:Tritium; D004099:Dihydroxyacetone Phosphate; D003903:Deuterium; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase