STUDYING THE CATALYTIC MECHANISM OF DETHIOBIOTIN SYNTHETASE USING KINETIC CRYSTALLOGRAPHY

Helena Käck¹, Katharine J. Gibson², Ylva Lindqvist¹ and Gunter Schneider¹.

¹ Division of Molecular Structural Biology, MBB; Karolinska Institutet, Stockholm, Sweden.
² DuPont Central Research and Development, Wilmington, USA.

Structural changes during the catalytic cycle of the ATP-dependent protein dethiobiotin synthetase (DTBS) has been studied using kinetic crystallography.

DTBS catalyses the conversion of 7,8-diaminopelargonic acid (DAPA) to dethiobiotin, the precursor of vitamin H. ATP and carbon dioxide are used as co-substrate and Mg²⁺ serves as co-factor. The reaction involves three different reaction intermediates, carbamylated DAPA, a carbamic-phosphoric mixed anhydride and tetrahedral intermediate. Our objective is to structurally characterise these intermediates using time-resolved crystallographic methods.

The structure of DTBS with the fist intermediate that forms on the enzyme in the absence of ATP has earlier been solved (1,2).

For trapping the second intermediate the reaction in the protein crystals was initiated in three different ways : (1) By adding substrate and ATP in a carbon dioxide free environment. The reaction is started by addition of CO₂ which quickly diffuses through the crystal. (2) By using conditions where the intermediate has been found to be semi-stable in the crystals, and (3) by using a photo-labile ATP analogue which is activated by light pulses.

All three ways have proven to be successful and resulted in the same structure of the phosphorylated intermediate.

Conditions for trapping the last intermediate has been found and crystallographic studies are underway.

1. Huang W, Jia J, Gibson K J, Taylor W S, Rendina A R, Schneider G & Lindqvist Y (1995) Biochemistry 36, 8474-8478.
2. Alexeev D, Baxter R L, Semkal O & Sawyer L (1995) Structure 3, 1207-1215.