Remarkable stabilization of zwitterionic intermediates may account for a billion-fold rate acceleration by thiamin diphosphate-dependent decarboxylases

When the E91D variant of ape-yeast pyruvate decarboxylase (EC 4.1.1.1) is e xposed to C2 alpha-hydroxybenzylthiamin diphosphate, this putative intermed iate is partitioned on the enzyme between release of the benzaldehyde produ ct (as evidenced by regeneration of active enzyme) and dissociation of the proton at C2 alpha to form the enamine-C2 alpha-carbanion intermediate. Whi le the pK(a) (the negative log of the acid dissociation constant) for this dissociation is similar to 15.4 in water, formation of the enamine at pH 6. 0 on the enzyme indicates a >9 unit pK(a) suppression by the enzyme environ ment. The dramatic stabilization of this zwitterionic enamine intermediate at the active center is sufficient to account for as much as a 10(9)-fold r ate acceleration on the enzyme. This "solvent" effect could be useful for a chieving the bulk of the rate acceleration provided by the protein over and above that afforded by the coenzyme on all thiamin diphosphate-dependent 2 -oxo acid decarboxylases.