Coupling of oxidation of substrates to reductive biosyntheses. III. Studies with L- and D-lactates.

In experiments employing hydrogen isotopes as tracers the possibility of relatively large isotope effects may be anticipated. In the main, this is because of the difference in zero-point energy between a bond to deuterium or tritium and the corresponding bond to hydrogen. When a hydrogen isotope effect is encountered in a rate process the result may be interpreted as evidence that the breaking of a bond to hydrogen is a rate-determining step in the reaction. Inasmuch as the formation of triosephosphate from lactate involves the breaking of a number of C-H bonds in oxidation reactions, it was deemed of interest to ascertain whether such a step plays a part in determining the rate of the over-all process. Furthermore, knowledge of the presence or absence of an isotope effect would be of aid in assessing quantitatively the extent of utilization of the a-hydrogen atom of lactate in the formation of the reduced product. For the purposes of the present studies we have compared the relative utilization of deuterium and tritium located in the (Yposition of I.-lactate in the synthesis of triosephosphate. In previous reports (1, 2) data were presented showing that liver glycogen formed from 2-D-Iclactate contains deuterium in the 4 and 6 positions of the glucosyl residues. These results were interpreted as evidence of coupling of the lactic and triosephosphate dehydrogenase systems and of the lactic dehydrogenase and malic enzyme systems. By administering together ~-T-Llactate and 2-D-L-lactate and measuring the specific activity of the tritium and the deuterium concentration in the 4 and 6 positions of the glucose units of the liver glycogen, it can be ascertained whether breaking of a C-H bond determines the rate synthesis of triosephosphate from lactate. The results of the present studies reveal that there is no discernible hydrogen isotope effect in the series of reactions leading from lactate to triosephosphate. From this it would appear that the rate-determining step in the formation of triosephosphate from lactate does not involve the breaking of C-H bonds. The amount of deuterium or tritium in liver glycogen formed from 2-Dor 2-T-Irlactate may be interpreted as a direct measure of the amount of hydrogen of exogenous lactate which is utilized in the reductive synthesis of carbohydrate.