Reaction Intermediate Analogues for Enolase

A number of compounds that appear to be analogues of the aci form of the normal carbanion intermediate are good inhibitors of yeast enolase. These include (3-hydroxy-2-nitropropyl)phosphonate (I), the ionized (pK = 8.1) nitronate form of which in the presence of 5 mM Mg2+ has a Ki of 6 nM, (nitroethyl)phosphonate (III) (pK = 8.5; Ki of the nitronate in the presence of 5 mM Mg2+ = 1 microM), phosphonoacetohydroxamate (IV) (pK = 10.2; Ki with saturating Mg2+ for the ionized form = 15 pM), and (phosphonoethyl)nitrolate (VII) (Ki at 1 mM Mg2+ = 14 nM). The oxime of phosphonopyruvate (VI) has a pH-independent Ki of 75 microM. I, IV, VI, and VII are slow binding inhibitors. All of these compounds are trigonal at the position analogous to C-2 of 2-phosphonoglycerate and contain a phosphono group, but a negatively charged metal ligand at the position isosteric with the hydroxyl attached to C-3 of 2-phosphoglycerate (as in IV) appears to contribute more to binding than a nitro group isosteric with the carboxyl of 2-phosphoglycerate (I and III). These data support the carbanion mechanism for enolase and suggest that the 3-hydroxyl of 2-phosphoglycerate is directly coordinated to Mg2+ prior to being eliminated to give phosphoenolpyruvate.