An Uncleavable Procaspase-3 Mutant Has a Lower Catalytic Efficiency but an Active Site Similar to That of Mature Caspase-3
| Autor: | A. Clay Clark, Brett Feeney, Kakoli Bose, Cristina Pop |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Conformational change Protein Conformation Proteolysis Biochemistry Catalysis Fluorescence Article Enzyme activator Zymogen medicine Trypsin Binding site DNA Primers chemistry.chemical_classification Enzyme Precursors Binding Sites Base Sequence medicine.diagnostic_test biology Caspase 3 Chemistry Hydrolysis Active site Enzyme Activation Enzyme Caspases biology.protein Biophysics medicine.drug |
| Zdroj: | Biochemistry. 42:12298-12310 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi034998x |
| Popis: | We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.5 and 25 degrees C the K(m) values are similar, the catalytic efficiency (k(cat)) is approximately 130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pK(a) values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5-9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2'. The pK(a) values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH. |
| Databáze: | OpenAIRE |
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