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Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism
| Content Provider | Scilit |
|---|---|
| Author | Ohshima, Toshihisa Sakane, Masaru Yamazaki, Takashi Soda, Kenji |
| Copyright Year | 1990 |
| Description | Journal: Journal of Biological Inorganic Chemistry Alanine dehydrogenase $(L‐alanine:NAD^{+}$ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation. red‐Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 °C for at least 60 min and with incubation in pH 5.5–9.5 at 75 °C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (– 20 °C, at pH 7.2) for over 5 months. The optimum pH for the L‐alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L‐alanine in the presence of $NAD^{+}$, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2‐oxobutyrate and 3‐fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L‐alanine then $NAD^{+}$. A dead‐end inhibition by the formation of an abortive ternary complex which consists of the enzyme, $NAD^{+}$ and pyruyate was included in the reaction. A possible role of the dead‐end inhibition is to prevent the enzyme from functioning in the L‐alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM: ammonia. 38.0 mM; L‐alanine, 10.5 mM and $NAD^{+}$, 0.26 mM. |
| Related Links | https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb19180.x |
| Ending Page | 720 |
| Page Count | 6 |
| Starting Page | 715 |
| ISSN | 09498257 |
| e-ISSN | 14321327 |
| DOI | 10.1111/j.1432-1033.1990.tb19180.x |
| Journal | Journal of Biological Inorganic Chemistry |
| Issue Number | 3 |
| Volume Number | 191 |
| Language | English |
| Publisher | Wiley-Blackwell |
| Publisher Date | 1990-08-01 |
| Access Restriction | Open |
| Subject Keyword | Journal: Journal of Biological Inorganic Chemistry Biotechnology and Microbiology |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry Inorganic Chemistry |
