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Observation and time resolution of chiral thiamin diphosphate‐bound intermediates in the catalytic cycle of pyruvate decarboxylase and benzoylformate decarboxylase by stopped‐flow circular dichroism.
Author(s) -
Chakraborty Sumit,
Jordan Frank,
Kneen Malea M.,
McLeish Michael J.,
Kenyon George L.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a40-c
Subject(s) - pyruvate decarboxylase , chemistry , decarboxylation , stereochemistry , thiamine , catalytic cycle , enamine , thiamine pyrophosphate , reaction intermediate , circular dichroism , catalysis , enzyme , cofactor , biochemistry , alcohol dehydrogenase
Yeast pyruvate decarboxylase (YPDC) and benzoylformate decarboxylase (BFD) are thiamin diphosphate (ThDP)‐dependent enzymes catalyzing the non‐oxidative decarboxylation of pyruvate and benzoylformate to give acetaldehyde and benzaldehyde, respectively. The ThDP‐bound intermediates implicated in the catalytic cycle are C2α‐lactylThDP (C2α‐mandelylThDP), the enamine/C2α‐carbanion and C2α‐hydroxyethylThDP (C2α‐hydroxybenzylThDP). Using circular dichroism (CD), both the C2α‐mandelyl‐ThDP type intermediate, and the enamine derived from the conjugated substrate analogue (E)‐2‐oxo‐4‐(3‐pyridyl)‐butenoic acid could be observed on YPDC and BFD. Especially intriguing is the ability to observe the enamine intermediate by CD, since only the imposed V conformation imparts chirality to it (no chiral centers). Time resolution of the intermediates was afforded by stopped‐flow UV and CD spectrometry. This study provided a novel view of the spatial orientation of ThDP‐bound intermediates in the enzyme environment as well as important mechanistic information of the catalytic cycle. Supported at Rutgers by NIH GM50380, and at Michigan by NSF EF‐0425719