Structure‐function studies of the regulatory bicarbonate binding in H. influenzae carbonic anhydrase: a functional reversion of allostery using multiple site mutations

Abstract

Type II β‐carbonic anhydrases are metalloenzymes that catalyze the conversion of carbon dioxide to bicarbonate using a characteristic closed four‐side chain coordination sphere for the active site Zn ion. Non‐catalytic bicarbonate binding sites have been discovered in some type II β‐CAs 8 Å away from the active site. A network of seven hydrogen bonds holds the inhibitory bicarbonate in place, and kinetic studies confirm cooperative inhibition by bicarbonate at sufficient levels. A comparison of bicarbonate‐bound HICA with type I β‐CAs lacking the non‐catalytic bicarbonate binding site reveals a number of principal interactions that might be responsible for the allosteric regulation of HICA. W39 provides a direct, stabilizing hydrogen bond to the inhibitory bicarbonate, a one‐residue shift of proline 48 changes an influential backbone orientation of the 44–48 loop, and a G41 residue makes additional room in the allosteric binding site compared to a conserved Ala in non‐allosteric β‐CAs. Individual site mutants of W39 have revealed predictable decreases in cooperativity consistent with the loss of a stabilizing hydrogen bond to the bicarbonate. Structure function studies of compound site mutants, however, abrogate the binding of inhibitory bicarbonate in the crystalline state even at high concentrations.This work is supported by NSF grant CHE‐0819686 and MCB‐0741396.