Biochemistry - Chemical Reactions in Living Cells, Volume 2 - D. Metzler 1980

Types of reactions catalyzed by enzymes
Enolic intermediates in isomerization reactions
Ketol-isomerases

Reactions Involving the oxidation of one functional group of a molecule by a neighboring group within the same molecule are characteristic of many metabolic pathways. In most cases, an enol is formed as an intermediate—either from ketones [equation (7-53)] or via dehydration [equation (7-59)]. One group of Enzymes catalyzes the interconversion of aldose sugars into their corresponding 2-ketoses (reaction 4.B in Table 7-1). Glucose-6-phosphate isomerase has been shown to operate with high efficiency in all Cells [130]. The enzyme from rabbit Muscle (a dimer with a Molecular Weight of 132,000) converts glucose-6-phosphate into fructose-6-phosphate with a turnover rate of ~10³ s^-1. The nonenzymatic equivalent of this and similar reactions is the base-catalyzed Lobry de Bruyn–Alberda van Ekenstein transformation [131], which was investigated in 1895 by two Dutch chemists after whom it was later named. In the same year, Emil Fischer suggested that an enediol serves as an intermediate in this reaction. The existence of the enediol has been confirmed by modern studies of the reaction mechanism.

When the isomerization of glucose-6-phosphate by glucose-6-phosphate isomerase is carried out in ²Н₂О, deuterium is found in the fructose-6-phosphate molecule at the C-1 position. This result is consistent with an enediol mechanism:

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It has been established that in the reverse reaction, the conversion rate of ²H-labeled fructose-6-phosphate is only 45% of that of the ¹H-containing compound. Thus, a primary deuterium kinetic isotope effect was observed, as expected for a rate-limiting C—H bond Cleavage.

A striking result was obtained when fructose-6-phosphate labeled with ²H and ¹⁴C at the 1-position underwent isomerization in the presence of a large excess of unlabeled fructose-6-phosphate. The glucose-6-phosphate reaction product contained not only ¹⁴C but also ²H, and isotope distribution analysis indicated that ²H is incorporated (at the C-2 position) via a direct transfer from the C-1 position [130]. This indicates that during half of the enzyme's catalytic turnovers, the ²H removed from the C-1 position is added back to the same molecule at the C-2 position. This intramolecular proton transfer points to a *syn* addition, i.e., a process in which the proton is abstracted and added from the same face of the molecule¹⁾. The occurrence of *syn* transfer, combined with the known configuration of glucose at C-2, indicates that the intermediate is a *cis*-enediol and that proton addition to either C-1 or C-2 of the enediol occurs from the *re* face. In every case investigated, other ketol isomerases also utilize a *cis*-enediol, and in most instances, addition occurs from the *re* face. However, mannose-6-phosphate isomerase catalyzes addition to the *si* face.

¹⁾ However, this argument should be treated with some caution. Nonenzymatic reactions are known in which a proton is abstracted and added from opposite faces of the molecule without any exchange with the solvent taking place [132].