Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B
| Autor: | Nam-Chul Ha, Bee Hak Hong, Kwan Yong Choi, Gyu Hyun Nam, Do Hyung Kim, Byung-Ha Oh, Young Sung Yun, Do Soo Jang |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Protein Denaturation Protein Folding Stereochemistry Equilibrium unfolding Steroid Isomerases Isomerase Crystallography X-Ray Biochemistry Catalysis Protein Structure Secondary chemistry.chemical_compound Ketosteroid Enzyme Stability Amino Acids Site-directed mutagenesis Molecular Biology Conserved Sequence Alanine chemistry.chemical_classification Binding Sites biology Pseudomonas putida Circular Dichroism Active site Hydrogen Bonding General Medicine biology.organism_classification Recombinant Proteins Amino acid Protein Structure Tertiary Kinetics Spectrometry Fluorescence chemistry Amino Acid Substitution biology.protein Thermodynamics Dimerization |
| Zdroj: | Journal of biochemistry. 134(1) |
| ISSN: | 0021-924X |
| Popis: | Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Delta(5)-3-ketosteroids at a rate of the diffusion-controlled limit. The dimeric interactions mediated by Arg72, Glu118, and Asn120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme. The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A. Equilibrium unfolding analysis revealed that the DeltaG(U)(H(2)O) values for the R72A, E118A, N120A, and E118A/N120A mutants were decreased by about 3.8, 3.9, 7.8, and 9.5 kcal/mol, respectively, relative to that of the wild-type enzyme. The interface mutations not only decreased the k(cat)/K(M) value by about 8- to 96-fold, but also increased the K(D) value for d-equilenin, a reaction intermediate analogue, by about 7- to 17.5-fold. The crystal structure of R72A determined at 2.5 A resolution and the fluorescence spectra of all the mutants indicated that the interface mutations altered the active-site geometry and resulted in the decreases of the conformational stability as well as the catalytic activity of KSI. Taken together, our results strongly suggest that the conserved interface residues contribute to stabilization and structural integrity of the active site in the dimeric KSI. |
| Databáze: | OpenAIRE |
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