Paired Carboxylic Acids in Enzymes and Their Role in Selective Substrate Binding, Catalysis, and Unusually Shifted p Values.

Cathepsin A (CatA, EC 3.4.16.5, UniProtKB P10619 ) is a human lysosomal carboxypeptidase. Counterintuitively, crystal structures of CatA and its homologues show a cluster of Glu and Asp residues binding the C-terminal carboxylic acid of the product or inhibitor. Each of these enzymes functions in an acidic environment and contains a highly conserved pair of Glu residues with side chain carboxyl group oxygens that are approximately 2.3-2.6 Å apart. In small molecules, carboxyl groups separated by ∼3 Å can overcome the repulsive interaction by protonation of one of the two groups. The p of one group increases (p ∼ 11) and can be as much as ∼6 pH units higher than the paired group. Consequently, at low and neutral pH, one carboxylate can carry a net negative charge while the other can remain protonated and neutral. In CatA, E69 and E149 form a Glu pair that is important to catalysis as evidenced by the 56-fold decrease in / in the E69Q/E149Q variant. Here, we have measured the pH dependencies of log(), log(), and log(/) for wild type CatA and its variants and have compared the measured p with calculated values. We propose a substrate-assisted mechanism in which the high p of E149 (>8.5) favors the binding of the carboxylate form of the substrate and promotes the abstraction of the proton from H429 of the catalytic triad effectively decreasing its p in a low-pH environment. We also identify a similar motif consisting of a pair of histidines in -formylglutathione hydrolase.