Phosphoryl transfer by a concerted reaction mechanism in UMP/CMP-kinase

The reaction mechanism of phosphoryl transfer catalyzed by UMP/CMP-kinase f rom Dictyostelium discoideum was investigated by semiempirical AM1 molecula r orbital computations of an active site model system derived from crystal structures that contain a transition state analog or a bisubstrate inhibito r. The computational results suggest that the nucleoside monophosphate must be protonated for the forward reaction while it is unprotonated in the pre sence of aluminium fluoride, a popular transition state analog for phosphor yl transfer reactions. Furthermore, a compactification of the active site m odel system during the reaction and for the corresponding complex containin g AIF(3) was observed. For the active site residues that are part of the LI D domain, conformational flexibility during the reaction proved to be cruci al. On the basis of the calculations, a concerted phosphoryl transfer mecha nism is suggested that involves the synchronous shift of a proton from the monophosphate to the transferred PO3-group. The proposed mechanism is thus analogous to the phosphoryl transfer mechanism in cAMP-dependent protein ki nase that phosphorylates the hydroxyl groups of serine residues.