title: Dual catalytic apparatus of the thiamin diphosphate coenzyme: acid-base via the 1',4'-iminopyrimidine tautomer along with its electrophilic role.
abstract: It was recently reported (Jordan, F.; Zhang, Z.; Sergienko, E. A. Bioorg. Chem. 2002, 30, 188-198) that addition to the E477Q active-center variant of yeast pyruvate decarboxylase of (a) pyruvate on a rapid-scan UV stopped-flow, or (b) acetaldehyde or benzoylformate on a circular dichroism (CD) instrument, generates a new band with lambda(max) near 300-310 nm. A chemical model demonstrated that the wavelength is appropriate to the 1',4'-iminopyrimidine tautomer of the 4'-aminopyrimidine ring in thiamin diphosphate. Herein, we report the formation of a new positive CD band centered at 305 nm when the Escherichia colipyruvate dehydrogenase complex first E1 subunit and its variants are exposed to phosphonolactylthiamin diphosphate, a stable analogue of the covalent adduct formed between the substrate pyruvate and the C2 atom of thiamin diphosphate. The behavior of this CD band, whether it suggests saturation of the enzyme by phosphonolactylthiamin diphosphate, or its very existence (the band is not seen with the E571A E1 variant, where E571 is hydrogen bonded to the N1' atom of the 4'-aminopyrimidine ring), as well as its position are consistent with its assignment to the 1',4'-imino thiamin diphosphate tautomer on the enzyme, chiral by virtue of its fixed V conformation. The mechanism of binding of phosphonolactylthiamin diphosphate closely resembles that of thiamin diphosphate itself.
authors.affiliation: Department of Chemistry and the Program in Cellular and Molecular Biodynamics at Rutgers, the State University, Newark, New Jersey 07102, USA. fjordan@newark.rutgers.edu
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Nemeria
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Yan
authors.forename: Palaniappa
authors.initials: P
authors.lastname: Arjunan
authors.forename: William
authors.initials: W
authors.lastname: Furey
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D011743:Pyrimidines / Q000737:chemistry*; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003067:Coenzymes; D011743:Pyrimidines; D011768:Pyruvate Dehydrogenase Complex; D013835:Thiamine Pyrophosphate