Reaction mechanisms of thiamin diphosphate enzymes: new insights into the role of a conserved glutamate residue.

Subsequent to the demonstration in the late 1950s of the catalytic power of the C2 anion/ylid of thiamin diphosphate, further convincing evidence was provided demonstrating that the 4'-aminopyrimidine group plays a vital role in activation of this cofactor. Structural evidence from several crystal structures of thiamin diphosphate-dependent enzymes emphasized the presence of a glutamate residue in hydrogen-bonding distance from N1' as a conserved element in these enzymes. The important role of this conserved glutamate in promoting C2-H ionization and activation of thiamin diphosphate was emphasized by site-directed mutagenesis studies. This role was further elaborated by spectroscopic studies of the 4'-aminopyrimidine-iminopyrimidine tautomerization. The low polarity of the environment of the protein-bound thiazolium is an additional factor in the stabilization of the C2 anion/ylid. The recently determined crystal structure and mutagenesis studies of glyoxylate carboligase, in which the position of the conserved glutamate is occupied by valine, now show that, for the multi-step reaction catalyzed by this enzyme, the advantages of accelerating the ionization of C2-H by re-introducing a carboxylate are outweighed by the apparent overstabilization of intermediates.