Reverse protonation is the key to general acid-base catalysis in enolase

The pH dependence of enolase catalysis was studied to understand how enolase is able to utilize both general acid and general base catalysis in each direction of the reaction at near-neutral pHs. Wild-type enolase from yeast was assayed in the dehydration reaction (2-phospho-D-glycerate --> phosphoenolpyruvate + H2O) at different pHs. E211Q, a site-specific variant of enolase that catalyzes the exchange of the alpha-proton of 2-phospho-D-glycerate but not the complete dehydration, was assayed in a H-1/H-2 exchange reaction at different pDs. Additionally, crystal structures of E211Q and E168Q were obtained at 2.0 and 1.8 Angstrom resolution, respectively. Analysis of the pH profile of k(cat)/K-Mg for wild-type enolase yielded macroscopic pK(a) estimates of 7.4 +/- 0.3 and 9.0 +/- 0.3, while the results of the pD profile of the exchange reaction of E211Q led to a pK(a) estimate of 9.5 +/- 0.1. These values permit estimates of the four microscopic pK(a)s that describe the four relevant protonation states of the acidibase catalytic groups in the active site. The analysis indicates that the dehydration reaction is catalyzed by a small fraction of enzyme that is reverse-protonated (i.e., Lys345-NH2, Glu211-COOH), whereas the hydration reaction is catalyzed by a larger fraction of the enzyme that is typically protonated (i.e., Lys345-NH3+, Glu211-COO-). These two forms of the enzyme coexist in a constant, pH-independent ratio. The structures of E211Q and E168Q both show virtually identical folds and active-site architectures (as compared to wild-type enolase) and thus provide additional support to the conclusions reported herein. Other enzymes that require both general acid and general base catalysis likely require reverse protonation of catalytic groups in one direction of the reaction.