Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

The Pyruvate Dehydrogenase Complexes: Structure-based Function and Regulation

“…A "proton wire" mechanism (62, 63) with a hydrated "tunnel" of acidic residues and water molecules was suggested to enable direct communication for proton shuttling between two ThDP N1Ј atoms. Experimental evidence of such a proton wire pathway was obtained for E1ec (43). No proton wire pathway was identified for E1h.…”

“…Steady-state kinetic and spectroscopic observations of ThDP-dependent enzymes supported communication between active center ThDPs manifested by "half-of-the site" reactivity (or alternating active-site mechanism) (42,43,59,60). Solution NMR evidence of nonequivalence of two active centers in E1h and E1ec was obtained from H/D exchange kinetics at C2-H of E1-bound ThDP (42,43) and by analysis of covalent ThDPbound intermediates in E1ec and E1h catalysis (54,61).…”

“…Solution NMR evidence of nonequivalence of two active centers in E1h and E1ec was obtained from H/D exchange kinetics at C2-H of E1-bound ThDP (42,43) and by analysis of covalent ThDPbound intermediates in E1ec and E1h catalysis (54,61). A "proton wire" mechanism (62, 63) with a hydrated "tunnel" of acidic residues and water molecules was suggested to enable direct communication for proton shuttling between two ThDP N1Ј atoms.…”

Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex

“…The homodimeric E. coli E1p catalyzes the rate-limiting step in the overall PDHc reaction (6), whereas structural and biochemical studies revealed the important regions and residues involved (7)(8)(9)(10)(11)(12). Two residues, Glu-571 and His-407, present in the E1p active site play important roles for the ThDP binding: reaction activation for Glu-571 and communication between E1p and E2p-lipoyl domains for His-407 (13).…”

“…In all previous x-ray structures of E. coli E1p, including the apoenzymes (ThDP-and Mg 2ϩ -free) and holoenzymes as well as variants with active center substitutions and complexes with the inhibitor thiamin 2-thiathiazolone diphosphate, and the reaction intermediate analogue 2-phosphonolactyl-ThDP, the N-terminal region residues 1-55 were totally disordered and not locatable (7)(8)(9)(10)(11)(12). Here and throughout this work we use the terms "ordered" and "disordered" only in the crystallographic sense (i.e.…”