The mechanism of glucose 6-phosphate–d-myo-inositol 1-phosphate cyclase of rat testis. The involvement of hydrogen atoms

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Abstract
Comparisonof the initial 3H/14C ratios in specifically labeled D-glucose 6-phosphates with the final ratios in myo-inositol produced by glucose 6-phosphate-D-myo-inositol 1-phosphate cyclase from rat testis showed that, during the conversion, the hydrogen atoms at C-1 and C-3 were fully retained, one hydrogen atom was lost from C-6, and that at C-5 was apparently retained to the extent of 80-90%. The loss of 3H could not be stimulated by addition of unlabeled NADH, and when unlabeled substrate was used % from [3H]NADH and [3H]water was not incorporated. Treatment of the enzyme with charcoal abolished the activity, and this was restored to 25-50% of the original activity by NAD+. The charcoal-treated enzyme again apparently gave 85% retention of hydrogen with [5-3H]glucose 6-phosphate as substrate in the presence of NAD+ alone, but the retention was decreased to 65% with excess of NADH. The results are interpreted as indicating that the cyclization proceeds by an aldol condensation in which C-5 is oxidized by NAD+ in a tightly-bound ternary complex, and that the apparent loss of 3H when untreated enzyme is used is due to an isotope effect. It is suggested that after treatment with charcoal some exchange of NADH with an external pool may take place.