Substrate binding and carboxylation by dethiobiotin synthetase--a kinetic and X-ray study.
BACKGROUND: The vitamin biotin is a ubiquitous prosthetic group of carboxylase and transcarboxylase enzymes. Biotin biosynthesis occurs by similar pathways in microorganisms and plants. The penultimate step in biotin biosynthesis, catalyzed by dethiobiotin synthetase (DTBS), involves a unique ATP-dependent N-carboxylation, resulting in formation of the ureido ring function of dethiobiotin. The first two steps of dethiobiotin formation, which is a complex, multistep enzymatic reaction, have been elucidated by a combination of X-ray crystallography and kinetic methods. RESULTS: The first step in catalysis by DTBS is the formation of an enzyme-substrate complex and the second is the enzymatic carboxylation of the bound substrate. Both steps are Mg2+ dependent. The kinetic constants in the presence and absence of Mg2+ have been measured and a set of X-ray structures determined at different stages of the reaction. The conformational changes in the active site of the enzyme, induced by Mg2+, substrate binding and substrate carboxylation, have been monitored crystallographically and are discussed. Sulfate ions bound to DTBS may mimic the behaviour of the alpha- and gamma-phosphates of ATP in Mg2+ binding and in the subsequent steps of the reaction. CONCLUSIONS: Mg2+ is an essential cation for both substrate binding and carbamate formation by DTBS, when sulfate is present. The conformational changes induced at the active site in the DTBS-substrate complex, when Mg2+ is present, are small yet highly significant and serve to optimize the interactions between substrate and enzyme. DTBS is active as a homodimer and the substrate-binding site straddles both monomers in the dimer. The carboxylation site is unambiguously identified as the N-7 amino group of the substrate, rather than the N-8 amino group, as previously suggested. The elongated nucleotide-binding loop (the P loop) binds both ATP and substrate in a manner which suggests that this feature may be of wider importance.[1]References
- Substrate binding and carboxylation by dethiobiotin synthetase--a kinetic and X-ray study. Alexeev, D., Baxter, R.L., Smekal, O., Sawyer, L. Structure (1995) [Pubmed]
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