title: Isolation and chemistry of the mixed anhydride intermediate in the reaction catalyzed by dethiobiotin synthetase.
abstract: 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.
authors.affiliation: DuPont Central Research and Development, Experimental Station, P.O. Box 80328, Wilmington, Delaware 19880-0328, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Gibson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000599:Amino Acids, Diamino / Q000737:chemistry / Q000378:metabolism; D000812:Anhydrides / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D002219:Carbamates / Q000737:chemistry / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation
chemical_list: D000599:Amino Acids, Diamino; D000812:Anhydrides; D002219:Carbamates; D010710:Phosphates; D002245:Carbon Dioxide; C004720:7,8-diaminopelargonic acid; D001710:Biotin; C004749:desthiobiotin; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020024:dethiobiotin synthetase