Carboxyl residues in the active site of human phenol sulfotransferase (SULT1A1)
| Autor: | C Wooldridge, Guangping Chen, D Zhang, J L York, C N Falany, Anna Radominska-Pandya, P A Rabjohn |
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| Rok vydání: | 2000 |
| Předmět: |
Models
Molecular Stereochemistry Molecular Sequence Data Phosphoadenosine Phosphosulfate Glutamic Acid Biochemistry Substrate Specificity Residue (chemistry) Humans Computer Simulation Estrogen Sulfotransferase Amino Acid Sequence Peptide sequence chemistry.chemical_classification Aspartic Acid Binding Sites biology Dose-Response Relationship Drug Active site Isoxazoles Hydrogen-Ion Concentration Arylsulfotransferase Amino acid Enzyme Activation Kinetics Enzyme chemistry Reagent biology.protein Mutagenesis Site-Directed Specific activity Indicators and Reagents |
| Zdroj: | Biochemistry. 39(51) |
| ISSN: | 0006-2960 |
| Popis: | The carboxyl-specific amino acid modification reagent, Woodward's reagent K (WK), was utilized to characterize carboxyl residues (Asp and Glu) in the active site of human phenol sulfotransferase (SULT1A1). SULT1A1 was purified using the pMAL-c2 expression system in E. coli. WK inactivated SULT1A1 activity in a time- and concentration-dependent manner. The inactivation followed first-order kinetics relative to both SULT1A1 and WK. Both phenolic substrates and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) protected against the inactivation, which suggests the carboxyl residue modification causing the inactivation took place within the active site of the enzyme. With partially inactivated SULT1A1, both V(max) and K(m) changed for PAPS, while for phenolic substrates, V(max) decreased and K(m) did not change significantly. A computer model of the three-dimensional structure of SULT1A1 was constructed based on the mouse estrogen sulfotransferase (mSULT1E1) X-ray crystal structure. According to the model, Glu83, Asp134, Glu246, and Asp263 are the residues likely responsible for the inactivation of SULT1A1 by WK. According to these results, five SULT1A1 mutants, E83A, D134A, E246A, D263A, and E151A, were generated (E151A as control mutant). Specific activity determination of the mutants demonstrated that E83A and D134A lost almost 100% of the catalytic activity. E246A and D263A also decreased SULT1A1 activity, while E151A did not change SULT1A1 catalytic activity significantly. This work demonstrates that carboxyl residues are present in the active site and are important for SULT1A1 catalytic activity. Glu83 and E134 are essential amino acids for SULT1A1 catalytic activity. |
| Databáze: | OpenAIRE |
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