The role of the allosteric B site in the fumarase reaction.

The role of a malate binding site in a concavity external to the more deeply situated active site has been a major mystery of the fumarase reaction. The malate, within 12 A of the active site, was bound by hydrogen bonds to two main-chain amides and to two basic residues, H129 and R126. Mutation of the His of this so-called B site of Escherichia coli fumarase had little effect on the overall initial rate kinetics of the enzyme, which has obscured an understanding of the critical role of the site.

Methylglyoxal synthetase, enol-pyruvaldehyde, glutathione and the glyoxalase system.

enol-Pyruvaldehyde (ePY or 2-hydroxypropenal, O=C(H)-C(OH)=CH(2)) a transient intermediate in the alkaline decomposition of the triosephosphates to methylglyoxal is now observed by UV and (1)H NMR spectroscopy as the immediate product of the methylglyoxal synthetase (MGS) reaction: dihydroxyacetone-P --> P(i) + ePY --> methylglyoxal (MG). Analysis of ePY formed from 1-(13)C- and (1R, 3S) -[1,3-(2)H]-DHAP establishes the stereochemical course of its formation by MGS. Its rate of ketonization is much too slow to be in the sequence required for the assay of MGS by coupling of the MG produced to glyoxalase I (Glx I): MG + glutathione (GSH) --> (S)-lactylglutathione (D-LG).