Paired Carboxylic Acids In Enzymes And Their Role In Selective Substrate Binding, Catalysis, And Unusually Shifted Pk(A) Values

Cathepsin A (CatA, EC 3.4.16.5, UniProtKB P10619) is a human lysosomal carboxypeptidase. Counter-intuitively, 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 angstrom apart. In small molecules, carboxyl groups separated by similar to 3 angstrom can overcome the repulsive interaction by protonation of one of the two groups. The pK(a) of one group increases (pK(a) similar to 11) and can be as much as similar to 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 k(cat)/K-m in the E69Q/E149Qvariant. Here, we have measured the pH dependencies of log(k(cat)), log(K-m), and log(k(cat)/K-m) for wild type CatA and its variants and have compared the measured pK(a) with calculated values. We propose a substrate-assisted mechanism in which the high pK(a) 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 pKa in a low-pH environment. We also identify a similar motif consisting of a pair of histidines in S-formylglutathione hydrolase.