The phosphoryl-transfer mechanism of Escherichia coli phosphoenolpyruvate carboxykinase from the use of AlF3

The mechanism of reversible transfer of the gamma -phosphate group of ATP b y Escherichia coli phosphoenolpyruvate carboxykinase (PCK) on to its substr ate is of great interest. It is known that metallofluorides are accurate an alogs of the transition state in the context of kinase mechanisms. Therefor e, two complexes of PCK, one with AlF3, Mg2+ and ADP (complex I), the other with AlF3, Mg2+, ADP and pyruvate (complex II) were crystallized. The X-ra y crystal structures of these two complexes were determined at 2.0 Angstrom resolution. The Al atom has trigonal bipyramidal geometry that mimics the transition state of phosphoryl transfer. The Al atom is at a distance of 2. 8 Angstrom and 2.9 Angstrom from an oxygen atom of the beta -phosphoryl gro up of ADP in complex I and II, respectively. A water molecule in complex I and an oxygen atom of the pyruvate in complex II are located along the axis of the trigonal bipyramid on the side opposite to the beta -phosphoryl oxy gen with respect to the equatorial plane, suggesting that the complexes are close mimics of the transition state. Along with the presence of positivel y charged species around the AlF3 moiety, these results indicate that phosp horyl transfer occurs via a direct displacement mechanism with associative qualities. (C) 2001 Academic Press.