Biological Mechanisms Involved in the Formation of Deoxysugars

Abstract

Abstract Studies with crystallized homogeneous enzyme permit formulation of the following reaction sequence to be catalyzed by thymidine diphosphate-d-glucose oxidoreductase. TDP-glucose + enzyme-NAD+ ⇄ TDP-4-ketoglucose + enzyme-NADH + H+ (1) TDP-4-ketoglucose → TDP-4-keto-5,6-glucoseen + H2O (2) TDP-4-keto-5,6-glucoseen + enzyme-NADH + H+ → TDP-4-keto-6-deoxyglucose + enzyme-NAD+ (3) A detailed examination of Reaction 1 was achieved by the use of TDP-6-deoxy-d-glucose, a close structural analogue of the substrate. Direct proof for the participation of enzyme-bound NAD+ in this reaction was provided by spectrophotometric observation of enzyme-NADH, as well as by tritium incorporation from TDP-6-deoxy-d-glucose specifically labeled at carbon 4 of the glucose moiety with tritium. Hydrogen transfer Reactions 1 and 3 mediated by enzyme-bound coenzyme were found to have B stereospecificity with respect to the nicotinamide moiety. Formation of enzyme-NADH was accompanied by a proportional decrease of enzymatic activity. Conversely, upon incubation of TDP-4-keto-6-deoxyglucose with enzyme-NADH, enzyme-NAD+ was obtained, resulting in restoration of enzymatic activity.