The Mechanism of Enzymatic Isomerization of Glucose 6-Phosphate to L-Myo-Inositol 1-phosphate

Summary

By the addition of NaB³H₄ to a system of homogeneous L-myo-inositol 1-phosphate synthase, glucose 6-phosphate, and NAD⁺, inosose-2 1-phosphate was trapped as a mixture of tritiated myo-inositol and scyllo-inositol. Based on the concentration of the cyclitols and the specific radioactivity of H₁ of glucitol produced by concomitant reduction of glucose 6-phosphate we estimate that 2 moles of inosose-2 1-phosphate are bound per mole of enzyme. In controls without enzyme or without substrate the cyclitols were unlabeled, showing that incorporation of tritium was related to the actively synthesizing system. Iditol and glucitol H₅, epimeric alditols representing 5-ketoglucose 6-phosphate, the other postulated intermediate, were not significantly labeled. We conclude that 5-ketoglucose 6-phosphate has no finite existence, its cyclization to inosose-2 1-phosphate occurring as quickly as its formation from glucose 6-phosphate. Since inosose-2 1-phosphate is sufficiently long-lived to be trapped by borotritide reduction, its reduction by putative NADH in the enzymatic reaction must be the rate limiting step, and thus the site of hydrogen isotope effects observed previously.