Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component -active centers-evidence for a 'direct pathway' between the 4' -aminopyrimidine N1' atoms

TitleCommunication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component -active centers-evidence for a 'direct pathway' between the 4' -aminopyrimidine N1' atoms
Publication TypeJournal Article
Year of Publication2010
AuthorsNemeria, NS, Arjunan P, Chandresakhar K, Mossad M, Tittmann K, Furey W, Jordan F
JournalJournal of Biological Chemistry
Pagination-
Date PublishedJanuary 27, 2010
Abstract

Kinetic, spectroscopic and structural analysis tested the hypothesis that a chain of residues connecting the 4-aminopyrimidine N1-atoms of thiamin diphosphates (ThDP) in the two active centers of the E. coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (E571, E235, E237) and R606 resulted in impaired binding of the second ThDP, once the first active center was filled suggesting a pathway for communication between the two ThDPs: (1) Steady-state kinetic and fluorescence quenching studies revealed that on E571A, E235A, E237A and R606A substitutions ThDP binding in the second active center was affected. (2) Analysis of the kinetics of thiazolium C2H/D exchange of enzyme bound ThDP suggests half-of-the sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for slow exchanging site indicating that only one of two active sites is filled with ThDP. (3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. (4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that E235 makes no direct contact with the cofactor. The role of the conserved E571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.

URLhttp://www.jbc.org/content/early/2010/01/27/jbc.M109.069179.abstract