Isolation and chemistry of the mixed anhydride intermediate in the reaction catalyzed by dethiobiotin synthetase

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 P-i. 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 [O-18]CO2 produced O-18-enriched DTB and P-i, as the existence of such an intermediate would require. Second, a moderately stable intermediate that could be labeled with either (CO2)-C-14, [gamma-P-33]ATP, [9-H-3]DAPA, or [1,7-C-14]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 P-i; under basic conditions, the intermediate cyclized, yielding DTB and P-i. When returned to fresh enzyme at pH 7.5, the intermediate underwent cyclization at a rate comparable to that of normal turnover.