The use of kinetic isotope effects in the study of metabolic control. I. Degradation of glucose-1-D by the hexosemonophosphate pathway.

The observation that molecules substituted with different isotopes may react at different rates has been of great value to the study of chemical reaction mechanisms. The theory and applications of this effect, particularly with isotopes of hydrogen, are well described in the literature (l-3), and frequent use is made of isotope effects in the study of the mechanism of enzyme reactions (4-6). One of the questions that is answered by the comparison of isotopically substituted molecules in a chemical or enzyme-catalyzed reaction is whether the component step concerned with the cleavage of the bond containing the isotope is rate-determining for the over-all reaction. For example, it was demonstrated recently (4) that in the conversion of dihydroxyacetone phosphate to n-glyceraldehyde-3-P by the enzyme triosephosphate isomerase, in which a C-H bond is broken and the proton released into the medium, the reaction proceeds at about half the rate if deuterium is present in the particular position in the substrate instead of hydrogen. This observation provides important information about the meaning of.the various kinetic constants measured for the isomerase reaction, and signifies that any mechanism formulated for the reaction must be consistent with the finding that the deprotonation of the substrate is slow relative to the other component steps of the reaction. By extension of this principle, it should be possible to determine whether, in a multi-enzyme sequence of reactions, a particular step is rate-limiting, depending upon whether isotope substitution at the bond broken in that reaction affects the rate of the whole sequence of reactions. For example, it may be asked whether in the conversion of glycerol to glycogen in the rat, the triosephosphate isomerase reaction is rate-determining. Since it is known that the isomerase reaction shows a deuterium isotope effect, this question could be approached by the use of glycerol containing deuterium which would be in the position labilized by isomerase when the glycerol is converted to dihydroxyacetone phosphate in the rat. The glycerol should be labeled with Cl4 so that its rate of incorporation into the glycogen could be conveniently determined.1 However, in preparing this doubly labeled glycerol, it is necessary that the Cl4 and deuterium be located in the same molecule, intramolecularly doubly labeled; otherwise, the Cl4 will not be a tracer for the deuterated species of glycerol. The complications that may arise from the use of a