Paired Carboxylic Acids in Enzymes and Their Role in Selective Substrate Binding, Catalysis, and Unusually Shifted pKa 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 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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2019 |
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| Erschienen: |
2019 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:58 |
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| Enthalten in: |
Biochemistry - 58(2019), 52 vom: 31. Dez., Seite 5351-5365 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Khavrutskii, Ilja V [VerfasserIn] |
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| Links: |
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| Themen: |
Carboxylic Acids |
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| Anmerkungen: |
Date Completed 22.06.2020 Date Revised 22.06.2020 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1021/acs.biochem.9b00429 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM298094665 |
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| 100 | 1 | |a Khavrutskii, Ilja V |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Paired Carboxylic Acids in Enzymes and Their Role in Selective Substrate Binding, Catalysis, and Unusually Shifted pKa Values |
| 264 | 1 | |c 2019 | |
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| 500 | |a Date Revised 22.06.2020 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, U.S. Gov't, Non-P.H.S. | |
| 650 | 7 | |a Carboxylic Acids |2 NLM | |
| 650 | 7 | |a Cathepsin A |2 NLM | |
| 650 | 7 | |a EC 3.4.16.5 |2 NLM | |
| 700 | 1 | |a Compton, Jaimee R |e verfasserin |4 aut | |
| 700 | 1 | |a Jurkouich, Kayla M |e verfasserin |4 aut | |
| 700 | 1 | |a Legler, Patricia M |e verfasserin |4 aut | |
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| 773 | 1 | 8 | |g volume:58 |g year:2019 |g number:52 |g day:31 |g month:12 |g pages:5351-5365 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1021/acs.biochem.9b00429 |3 Volltext |
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