| The PDHc-E1 catalyzed decarboxylation of pyruvate involves two unstable zwitterionic intermediates-the C2-carbanion/ylide/carbene and the C2alpha-carbanion or enamine. The enzyme's effect in the stabilization of the unstable intermediate was examined using HEThDP-d4 (C2alpha-hydroxylethylthiamin diphosphate which carries one deuteron at the C2alpha and three deuterons at the C2beta positions) as the model compound. The deuterium abstraction rate of the C2alpha-D bond was determined, and the acceleration by the PDHc-E1 "environmental" effect discussed. It was concluded that the PDHc-E1 accelerates enamine formation by at least a factor of 107. This factor corresponds to a 10 kcal/mol lowering of the activation energy barrier.; The crystal structure of PDHc-E1 from E. coli reveals no interpretable electron density for the N-terminal 1-55 amino acids. Mass spectroscopic evidence showed that the N-terminal region plays a major role in the interaction with the PDHc-E2 subunit. MS data also showed that the residues 222-367 in PDHc-E1 are protected by PDHc-E2, suggesting recognition of the active site.{09}There is also evidence to suggest that the conformation of the cleft, acting as a gating mechanism to control the influx and efflux of substrates/solvents, is at least partially regulated by ThDP.; Two PDHc-E1 mutants were constructed with single amino acid substitution at histidine 407 and glutamate 636, respectively. Comparison of their peptide maps with that of the parental protein revealed changes in the mobility of loops such as 240-247, 401-413 and 541-557, all of them are adjacent to the active binding site. FTICR MS data also suggest that both the N-terminus and C-terminus can be affected by the mutation.; We showed that the amino acid residues from the ThDP-binding fold are not part of the Mab18A9 binding epitope. Rather, the amino acid residues 157-181, 383-410 and 541-557 are part of the epitope for MAb18A9 binding. A common structural feature of these protected peptides is that they all exhibit randomness in their secondary structure. It is hypothesized that binding of these peptides by Mabl8A9 blocks the pathway responsible for the influx and efflux of substrate. |
