Exchange reactions catalyzed by group-transferring enzymes oppose the quantitation and the unravelling of the identify of the pentose pathway.
1. The distributions and rates of transfer of carbon isotopes from a selection of specifically labelled ketosugar-phosphate substrates by exchange reactions catalyzed by the pentose and photosynthetic carbon-reduction-pathway group-transferring enzymes transketolase, transaldolase and aldolase have been measured using 13C-NMR spectroscopy. 2. The rates of these exchange reactions were 5, 4 and 1.5 mumol min-1 mg-1 for transketolase exchange, transaldolase exchange and aldolase exchange, respectively. 3. A comparison of the exchange capacities contributed by the activities of these enzymes in three in vitro liver preparations with the maximum non-oxidative pentose pathway flux rates of the preparations shows that transketolase and aldolase exchanges exceeded flux by 9-19 times in liver cytosol and acetone powder enzyme preparations and by 5 times in hepatocytes. Transaldolase was less effective in the comparison of exchange versus flux rates: transaldolase exchange exceeded flux by 1.6 and 5 in catalysis by liver cytosol and acetone powder preparations, respectively, but was only 0.6 times the flux in hepatocytes. 4. Values of group enzyme exchange and pathway flux rates in the above three preparations are important because of the feature role of liver and of these particular preparations in the establishment, elucidation and measurement of a proposed reaction scheme for the fat-cell-type pentose pathway in biochemistry. 5. It is the claim of this paper that the excess of exchange rate activity (particularly transketolase exchange) over pathway flux will overturn attempts to unravel, using isotopically labelled sugar substrates, the identity, reaction sequence and quantitative contribution of the pentose pathway to glucose metabolism. 6. The transketolase exchange reactions relative to the pentose pathway flux rates in normal, regenerating and foetal liver, Morris hepatomas, mammary carcinoma, melanoma, colonic epithelium, spinach chloroplasts and epididymal fat tissue show that transketolase exchange may exceed flux in these tissues by factors ranging over 5-600 times. 7. The confusion of pentose pathway theory by the effects of transketolase exchange action is illustrated by the 13C-NMR spectrum of the hexose 6-phosphate products of ribose 5-phosphate dissimilation, formed after 30 min of liver enzyme action, and shows 13C-labelling in carbons 1 and 3 of glucose 6-phosphate with ratios which range over 2.1-6.4 rather than the mandatory value of 2 which is imposed by the theoretical mechanism of the pathway.
Flanigan
I
I
Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra.
Collins
J G
JG
Arora
K K
KK
MacLeod
J K
JK
Williams
J F
JF
D000818:Animals; D002384:Catalysis; D002478:Cells, Cultured; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010429:Pentoses / Q000378:metabolism*; D010944:Plants; D051381:Rats; D011915:Rats, Inbred BUF; D014153:Transaldolase / Q000378:metabolism*; D014174:Transketolase / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D010429:Pentoses; D014174:Transketolase; D014153:Transaldolase; D005634:Fructose-Bisphosphate Aldolase