Identification of Significant Residues in the Substrate Binding Site of Bacillus stearothermophilus Farnesyl Diphosphate Synthase
| Autor: | Takashi Doi, Ayumi Koike-Takeshita, Masaya Tajima, Hiroaki Sano, Kyozo Ogura, Tokuzo Nishino, Shusei Obata, Tanetoshi Koyama |
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| Rok vydání: | 1996 |
| Předmět: |
Indoles
Stereochemistry Acyclic Monoterpenes Molecular Sequence Data Lysine Biochemistry Geobacillus stearothermophilus Hemiterpenes Organophosphorus Compounds Farnesyl diphosphate synthase Transferases Amino Acid Sequence Enzyme kinetics Binding site Conserved Sequence Alanine chemistry.chemical_classification Alkyl and Aryl Transferases Binding Sites Base Sequence Molecular Structure biology Terpenes Wild type Geranyltranstransferase Farnesol Amino acid Kinetics Models Chemical chemistry Mutagenesis Site-Directed biology.protein Chromatography Thin Layer Diterpenes Isoleucine Sequence Alignment |
| Zdroj: | Biochemistry. 35:9533-9538 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi960137v |
| Popis: | Farnesyl diphosphate synthases have been shown to possess seven highly conserved regions (I-VII) in their amino acid sequences [Koyama et al. (1993) J. Biochem. (Tokyo) 113, 355-363]. Site-directed mutants of farnesyl diphosphate synthase from Bacillus stearothermophilus were made to evaluate the roles of the conserved aspartic acids in region VI and lysines in regions I, V, and VI. The aspartate at position 224 was changed to alanine or glutamate (mutants designated as D224A and D224E, respectively); aspartates at positions 225 and 228 were changed to isoleucine and alanine (D225I, D228A); lysine at position 238 was changed to either alanine or arginine (K238A, K238R). The lysines at positions 47 and 183 were changed to isoleucine and alanine (K471, K183A), respectively. Kinetic analyses of the wild-type and mutant enzymes indicated that the mutagenesis of Asp-224 and Asp-225 resulted in a decrease of Kcat values of approximately 10(4)- to 10(5)-fold compared to the wild type. On the other hand, D228A showed a Kcat value approximately one-tenth of that of the wild type, and the k(m) value for isopentenyl diphosphate increased approximately 10-fold. Both K471 and K183A showed k(m) values for isopentenyl diphosphate 20-fold larger and kcat values 70-fold smaller than the wild type. These results suggest that the two conserved lysines in regions I and V contribute to the binding of isopentenyl diphosphate and that the first and the second aspartates in region VI are involved in catalytic function. Aspartate-228 is also important for the binding of isopentenyl diphosphate rather than for catalytic reaction. |
| Databáze: | OpenAIRE |
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