Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase.
The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with lambda(max) at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with lambda(max) at 437 nm. The time course of formation/depletion of the PKB-ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB-ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with lambda(max) at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 A) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2alpha atom, rather than of the enamine, and supporting the assignment of the lambda(max) at 473 nm to the PAA-ThDP adduct. The structure of the PAA-ThDP covalent complex is the first example of a product-ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.
Chakraborty
Sumit
S
Department of Chemistry, Rutgers University, Newark, New Jersey 07102, USA.
Nemeria
Natalia S
NS
Balakrishnan
Anand
A
Brandt
Gabriel S
GS
Kneen
Malea M
MM
Yep
Alejandra
A
McLeish
Michael J
MJ
Kenyon
George L
GL
Petsko
Gregory A
GA
Ringe
Dagmar
D
Jordan
Frank
F
D001665:Binding Sites; D002087:Butyrates / Q000737:chemistry* / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D003653:Decarboxylation; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology*; D011725:Pyridines / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
C529114:2-oxo-4-(pyridin-3-yl)-3-butenoic acid; D002087:Butyrates; D005456:Fluorescent Dyes; D011725:Pyridines; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase; D013835:Thiamine Pyrophosphate