Isolation and Chemistry of the Mixed Anhydride Intermediate in the Reaction Catalyzed by Dethiobiotin Synthetase
| Autor: | Katharine J. Gibson |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Stereochemistry
Kinetics Biotin Cleavage (embryo) Biochemistry Anhydrides Phosphates Catalysis Ligases chemistry.chemical_compound Adenosine Triphosphate Escherichia coli Organic chemistry Carbon-Nitrogen Ligases Phosphorylation chemistry.chemical_classification Quenching (fluorescence) Molecular Structure Chemistry Amino Acids Diamino Carbon Dioxide Amino acid Enzyme Urea Acid hydrolysis Carbamates Chromatography Thin Layer |
| Zdroj: | Biochemistry. 36:8474-8478 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi970447t |
| Popis: | Dethiobiotin synthetase (DTBS) catalyzes the formation of the cyclic urea, dethiobiotin (DTB), from (7R,8S)-diaminononanoic acid (DAPA), CO2, and ATP; the other products of the reaction are ADP and Pi. The first intermediate in the reaction sequence is the 7-carbamate of DAPA [Huang, W., et al. (1995) Biochemistry 34, 10985-10995; Gibson, K. J., et al. (1995) Biochemistry 34, 10976-10984; Alexeev, D., et al. (1995) Structure 3, 1207-1215]. The existence of the second postulated intermediate, a mixed carbamic-phosphoric anhydride formed when the carbamate is phosphorylated by ATP, is consistent with the cleavage of the gamma-phosphoryl group of ATP seen in DTBS reaction mixtures [Baxter, R. L., & Baxter, H. C. (1994) J. Chem. Soc., Chem. Commun., 759-760]. Two more direct lines of evidence for the mixed anhydride intermediate have now been obtained. First, a DTBS reaction mixture containing [18O]CO2 produced 18O-enriched DTB and Pi, as the existence of such an intermediate would require. Second, a moderately stable intermediate that could be labeled with either 14CO2, [gamma-33P]ATP, [9-3H]DAPA, or [1,7-14C]DAPA was trapped by quenching DTBS reactions at pH 4 and isolated by thin-layer chromatography. As expected for the proposed mixed anhydride, this species underwent acid hydrolysis to DAPA, CO2, and Pi; under basic conditions, the intermediate cyclized, yielding DTB and Pi. When returned to fresh enzyme at pH 7.5, the intermediate underwent cyclization at a rate comparable to that of normal turnover. |
| Databáze: | OpenAIRE |
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