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)-TH)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.
- Research Organization:
- Harvard Univ., Cambridge, MA (USA)
- OSTI ID:
- 6515664
- Journal Information:
- Biochemistry; (United States), Vol. 27:16
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ISOMERASES
BIOCHEMICAL REACTION KINETICS
CHICKENS
DEUTERIUM
ESCHERICHIA COLI
ISOTOPE EFFECTS
PROTONS
SACCHAROMYCES CEREVISIAE
THERMODYNAMICS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMALS
BACTERIA
BARYONS
BIRDS
ELEMENTARY PARTICLES
ENZYMES
EUMYCOTA
FERMIONS
FOWL
FUNGI
HADRONS
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
PLANTS
REACTION KINETICS
SACCHAROMYCES
STABLE ISOTOPES
VERTEBRATES
YEASTS
550201* - Biochemistry- Tracer Techniques