Role of conserved Met112 residue in the catalytic activity and stability of ketosteroid isomerase
| Autor: | Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Jae-Hee Jeong, Eun Ju Shin, Young Sung Yun, Do Soo Jang |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Stereochemistry Biophysics Steroid Isomerases Isomerase Biochemistry Catalysis Analytical Chemistry Enzyme catalysis 03 medical and health sciences chemistry.chemical_compound Methionine Ketosteroid Catalytic Domain Organic chemistry Transition Temperature Enzyme kinetics Amino Acid Sequence Molecular Biology Alanine 030102 biochemistry & molecular biology biology Hydrogen bond Pseudomonas putida Active site Hydrogen Bonding biology.organism_classification Ketosteroids 030104 developmental biology chemistry Amino Acid Substitution Mutation biology.protein Hydrophobic and Hydrophilic Interactions Sequence Alignment |
| Zdroj: | Biochimica et biophysica acta. 1864(10) |
| ISSN: | 0006-3002 |
| Popis: | Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core. |
| Databáze: | OpenAIRE |
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