Applications in Mechanistic Biochemistry

Most of the sugars specifically labeled with isotopes of hydrogen that have been synthesized were prepared for use in mechanistic studies of enzymes. 

In the course of enzyme reactions, carbon-bound hydrogen may exchange between the substrate and a cofactor, an enzyme, the solvent, or a second substrate molecule. When the hydrogen is transferred to a cofactor, it may either be retained, retransferred to the same or another position on the original substrate molecule, or transferred to another molecule. Usually, transfer to a cofactor, often a pyridine nucleotide, occurs as hydride, and transfer to an enzyme 

Enzymes catalyzing the same reaction but obtained from different sources may have very different properties, but usually operate by the same mechanism, an exception being D-fructose 1,6-diphosphate lyase (aldolase). Caution must, therefore, be observed in correlating mechanistic evidence derived for enzymes from different sources. 

Enzymes thus far found to catalyze isomerization or epimerization by oxidation and reduction require the cofactor NAD, which is often very tightly bound, not being removed by dialysis, but only by treatment with charcoal. Enzymes for which a keto-enol mechanism has been suggested do not usually involve NAD. The two mechanisms should also be distinguishable by the nonoccurrence or occurrence, respectively, of hydrogen exchange with the solvent. 

When D-glucose 6-phosphate and D-fructose 6-phosphate are inter-converted by D-glucose 6-phosphate ketol isomerase in either deuterium oxide33 or water-1 (Ref. 34), isotope is incorporated at C-l of D-fructose 6-phosphate and C-2 of D-glucose 6-phosphate, indicating that the interconversion involves an enediol intermediate, which may arise from an open-chain (37) or cyclic (38) form of the sugar, 

---