Partial reactions of D-glucose 6-phosphate-1L-myoinositiol 1-phosphate cyclase.
After removal of tightly bound NAD(+) by using charcoal, a preparation of d-glucose 6-phosphate-1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-(3)H]NADH to give [5-(3)H]-glucitol 6-phosphate and [5-(3)H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [(3)H]myoinositol 1-phosphate in the presence of [4-(3)H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH(4) in the presence of NAD(+). Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD(+). There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15x10(5).
D000818:Animals; D002238:Carbohydrate Epimerases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D003500:Cyclization; D005958:Glucosephosphates / Q000032:analysis / Q000138:chemical synthesis; D007294:Inositol; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009243:NAD; D011485:Protein Binding; D051381:Rats; D013402:Sugar Alcohols; D013737:Testis / Q000201:enzymology; D013997:Time Factors; D014316:Tritium
D005958:Glucosephosphates; D013402:Sugar Alcohols; D009243:NAD; D014316:Tritium; D007294:Inositol; D002238:Carbohydrate Epimerases