Observation of thiamin-bound intermediates and microscopic rate constants for their interconversion on 1-deoxy-D-xylulose 5-phosphate synthase: 600-fold rate acceleration of pyruvate decarboxylation by D-glyceraldehyde-3-phosphate.

The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-hydroxyethyl donor with d-glyceraldehyde-3-phosphate (d-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of D-GAP, while addition of D-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and D-GAP bound, and the central enzyme-bound enamine reacts with D-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of D-GAP suggests a new approach to inhibitor design.