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Paired Carboxylic Acids in Enzymes and Their Role in Selective Substrate Binding, Catalysis, and Unusually Shifted pKa Values
Version 2 2019-12-13, 18:07
Version 1 2019-07-22, 13:07
journal contribution
posted on 2019-12-13, 18:07 authored by Ilja V. Khavrutskii, Jaimee R. Compton, Kayla M. Jurkouich, Patricia M. LeglerCathepsin 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 pKa of one group increases (pKa ∼ 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 kcat/Km in the E69Q/E149Q variant. Here, we have measured the pH dependencies
of log(kcat), log(Km), and log(kcat/Km) for wild type CatA and its variants and have compared
the measured pKa with calculated values.
We propose a substrate-assisted mechanism in which the high pKa 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.