An Active-Site Bro̷nsted Acid-Base Catalyst Destabilizes Mandelate Racemase and Related Subgroup Enzymes: Implications for Catalysis.

Abstract

Enzymes of the enolase superfamily (ENS) are mechanistically diverse, yet share a common partial reaction, i.e., the metal-assisted, Bro̷nsted base-catalyzed abstraction of the α-proton from a carboxylate substrate to form an enol(ate) intermediate. Although the catalytic machinery responsible for the initial deprotonation reaction has been conserved, divergent evolution has led to numerous ENS members that catalyze different overall reactions. Using differential scanning calorimetry, we examined the contribution of the Bro̷nsted acid-base catalysts to the thermostability (T_m) of four members of the mandelate racemase (MR)-subgroup of the ENS: MR, d-tartrate dehydratase, l-talarate/galactarate dehydratase, and l-fuconate dehydratase. Each enzyme contains an active-site Lys (part of a KxK motif) and His, which act as Bro̷nsted acid-base catalysts. The KxK → KxM substitutions increased the thermostability in all four enzymes with the effect being most prominent for MR (ΔT_m = +8.6 °C). The KxK → MxK substitutions decreased the thermostability in all four enzymes, and the His → Asn substitution had a significant stabilizing effect only on MR. Thus, the active sites of MR-subgroup enzymes are destabilized by the Lys Bro̷nsted acid-base catalyst, suggesting that the destabilization energy may be used to drive a conformational change of the enzyme to yield a catalytically competent protonation state upon substrate binding.