TY - JOUR
T1 - Observation of thiamin-bound intermediates and microscopic rate constants for their interconversion on 1-deoxy- d -xylulose 5-phosphate synthase
T2 - 600-Fold rate acceleration of pyruvate decarboxylation by d -glyceraldehyde-3-phosphate.
AU - Patel, Hetalben
AU - Nemeria, Natalia S.
AU - Brammer, Leighanne A.
AU - Freel Meyers, Caren L.
AU - Jordan, Frank
PY - 2012/11/7
Y1 - 2012/11/7
N2 - 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.
AB - 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.
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U2 - 10.1021/ja307315u
DO - 10.1021/ja307315u
M3 - Article
C2 - 23072514
AN - SCOPUS:84868531635
SN - 0002-7863
VL - 134
SP - 18374
EP - 18379
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 44
ER -